CLINICAL CHEMISTRY 1

INTRODUCTION,REVIEW OF
BASIC CHEMISTRY AND LAB
MATHEMATICS

Shirley O.Solitario,RMT
 CLINICAL CHEMISTRY
 “clinical”- comes from the greek word kline,
meaning “bed”.
 “chemistry” –the science that deals with the
elements, their compounds and the chemical
structure and interaction of matter.
 Is a basic science that utilizes the specialty of
chemistry to study human beings in various
stages of health and disease.
 It is an applied science when analyses are
performed on body fluids or tissue specimens to
provide important information for the diagnosis or
treatment of disease.
 WHAT IS CLINICAL CHEMISTRY?
 Study of biochemical processes associated with
health and disease
 Measurement of constituents in the body fluids or
tissues to facilitate diagnosis of disease.
 Monitor the effect of treatment and measuring
the drug levels in blood and other body fluids.
 HISTORICAL BACKGROUND
 -Robert James Graves (1796-1853)- few and scanty,
indeed are the rays of light which chemistry has flung
on the vital mysteries”
 Johann Joseph Scherer (1814-1869)- first used the
term “clinical chemistry laboratory”( klinisch-
chemischen Laboratorium.)
 Max Josef von Pettenkofer (1818-1901)- complained
that clinicians do not use their chemistry laboratory
services except when needed for luxurious
embelishment for a clinical lecture.”
 HISTORICAL BACKGROUND
 FRIEDRICH WOHLER-synthesized urea during
early 1800.
 Diagnosis of DM by analysis of urine sugar dates
back to the middle ages.
 19th century

 The composition of starch and fats were known.

 A number of proteins from the blood has been

isolated and characterized
 HISTORICAL BACKGROUND
 Cholesterol was shown to be present in gallstone.
 Chemical make-up of urine was elucidated.
 First clinical chemistry text was written in 1836.
 IMPROVEMENTS IN
INSTRUMENTATION
 INVENTION OF pH METER
 First device to measure acidity of citrus fruits.

 Control of pH in enzyme assay and explore

effect of pH on the rate of reaction
 Measurement of blood pH.

• INVENTION OF COLORIMETER

 DR.LEONARD SKEGGS- developed a device

that would automatically pipette a sample,
add reagents to it, mix and incubate the
resulting solution and measure the color
change with a colorimeter.
CLINICAL CHEMISTRY BASICS
 Laboratory Mathematics, Calculations and
Statistical Concept
DEFINITION OF TERMS:
 SOLUTION- composed of solvent and solute.
In a solution, there is an even distribution of
molecules or ions throughout the solvent.
 Solute – refers to the substance being
dissolved which maybe a solid, liquid or gas.
 Solvent – refers to the substance in which the
solute is dissolved which most cases are
liquids.
1. CONCENTRATION
 refers to the weight or volume of the solute
present in a specified amount of the solvent or a
solution
THREE BASIC TYPES OF SOLUTIONS
 Percent solution
 amount of solute in a solution can be measured as a
percentage of the total volume of the solution
 percent refers to the parts of the solute per 100
parts of the solvent, represented by the percent sign
(%)
 3 EXPRESSIONS OF PERCENT SOLUTION
1. % weight per volume (w/v)- most common
2. % volume per volume (v/v)
3. % weight per weight (w/w)
 PERCENT SOLUTION
 A. weight/volume
 FORMULA:
% w/v= grams of solute/ mL of solution x 100
Where as:
grams of solute = % soln desired x total vol.desired
100
Example:
1. What amount of NaCl is needed to make 800ml of
0.85 % solution?
 Example:
1. What amount of NaCl is needed to make 800ml
of 0.85 % solution?
 GIVEN: TV= 800ml , % w/v= 0.85
 PROBLEM: GRAMS OF SOLUTE=?

SOLUTION:
grams of solute = % soln desired x total vol.desired
100
Grams of solute(NaCl)= 0.85 x 800
100
= 6.8 grams
6.8 grams of NaCl is required to make 800mL of
0.85% NaCl
 PERCENT SOLUTION
 B. % volume per volume (v/v)
 FORMULA: % v/v = mL of solute x 100

 mL of solution
 Where as:

 mL of solute= % soln.desired x total vol. desired

 100
 EXAMPLE FOR V/V
 Prepare 500mL 40% alcohol from stock absolute
alcohol solution.
 Given: tv= 500mL, %= 40%

 Problem: vol. of solute=?

 Solution:

 mL of solute= % soln.desired x total vol. desired

 100
 mL of solute = 40 x 500 / 100
 = 200 mL
 = 200mL alcohol + 300mL distilled water to make 500
mL solution.
 PERCENT SOLUTION
 % weight per weight (w/w)
 Formula:
 % w/w = grams of solute x 100
 grams of solution
 Where as:
 Grams of solute= % soln.desired x grams of total soln.
 100
 Note: When preparing concentrated acid
solutions, always add acid to water.
SAMPLE PROBLEM FOR W/W
 Make 1800mL of a 50% weight/weight acetone alcohol
solution.
 Specific gravity acetone: 0.786
 Specific gravity alcohol: 0.810
- Since you are working with liquids, the first step is to know
how many mL of each you will need to get 50 grams of each
to make a total 100 grams
- 50 = 63.66 mL of acetone required to get 50g
0.786
- 50 = 61.73 mL of alcohol required to get 50 g
0.810
 . The second step is to add the mL of acetone and
alcohol together. This tells you how many total Ml
are required to have 50 g of each substance
 63.60 mL of acetone
+ 61.73 mL of alcohol
125.33 mL total mL
Since the total volume needed is 1800, you need to
find how many 125.33 are in 1800mL. This is
accomplished by dividing 125.33 into 1800
1800/ 125.33 = 14.36
 The final step is to find the total volumes of
acetone and alcohol needed to make 1800mL of
the 50% solution.

 63.6mL of acetone x 14.36 = 913.296 total vol.acetone

 61.73mL of alcohol x 14.36 = 886.44 total vol .alcohol
 1799.74 mL total vol. soln.
 THREE BASIC TYPES OF SOLUTIONS
 Molar Solutions (MOLARITY)
 solution containing one gram molecular weight (one mole of
the solute in one liter solution) of the substance per liter of
the solution
 The number of moles expressed per 1 liter of solution.
 It is expressed as moles per liter (mol/L)
 FORMULA:
 mole = grams of solute
 molecular weight (MW)
MOLARITY(M)= grams of solute
MW x volume of solution (L)
 SAMPLE PROBLEM 1 (MOLARITY)

 If you wish to make 1 liter of 2M NaCl solution, how much

NaCl must be weighed?
 GMW of NaCl= 58.5 g MW Na (23) + MW Cl
(35.5)
 GMW- is obtained by adding the atomic weights
of the components elements.
 Formula:
 Grams of NaCl=M x MW x volume of solution(L)
 = 2 x 58.5 x 1
 = 117 g NaCl needed for 1L 2M Nacl solution.
SAMPLE PROBLEM 2 (MOLARITY)
 Prepare 250 mL of a 4.8 M solution of HCl
 GMW OF HCL=36.5gMW H(1) +MW Cl(35.5)
 Grams of HCl= M x MW x vol.of soln(L)
 = 4.8 x 36.5 x 0.250
 = 43.8 grams
 Dissolve 43.8 g HCl in distilled water to make
250mL of 4.8 M soln. of HCL
 Since L is required you have to convert the
given volume which is mL into liter
 1 liter = 1000ml = 250ml = 0.250
1000ml
 SAMPLE PROBLEM 3 (MOLARITY)
 A solution contains 24g NaOH per liter, what is the
molarity?
 GMW= 40g MW Na(23) + MW O(16) + MW H
(1)
 M = grams of solute

 MW x volume (L)
 M = 24__

40 x 1
M = 0.6
 SAMPLE PROBLEM 4(MOLARITY)
 How many mL of phosphoric acid is needed to prepare
1L of 2M solution of phosphoric acid?
 Note: since it is liquid, you need to account for the
specific gravity. Also since it is an acid, you need to
account for the % purity
 GMW H3 PO4= 97.9938g

 Specific gravity = 1.5 g/mL

 % purity = 85.5 %

 mL H3PO4= molarity x GMW x V (L)

 specific gravity x % purity

 = 2 x 97.9938 x 1
1.5 x 0.855
= 152.817 mL of acid diluted to 1000mL
 CONVERSION OF % W/V TO M

To convert % w/v to Molarity

(M)
M = % w/v x 10
GMW
THREE BASIC TYPES OF SOLUTIONS
 Normal Solutions (NORMALITY)
 one gram equivalent weight of solute in one liter of
solution
 Gram equivalent weight is that weight in grams of an
element or compound which will combine with or
replace one gram of hydrogen
 Gram equivalent weight of a substance is equal to
the gram molecular weight of a substance divided by
it, total positive or negative valence
 It has often been used in acid-base calculations.
 NORMALITY

 FORMULAS:
 Equivalent weight (EW)= molecular weight

valence
 Normality (N) = grams of solution

equivalent weight x volume (L)

SAMPLE PROBLEM 1 (NORMALITY)
If a solution contains 111g CaCl2 per liter, what is the
normality?
 MW CaCl2 = 111g MW Ca(40) + MW Cl (35.5) x 2

 Valence Ca = +2 EW = 111/ 2 = 55.5 g

 N = grams of solute

 EW x vol (L)
 = 111__
 55.5 x 1
 = 2 N CaCl2
 SAMPLE PROBLEM 2
(NORMALITY)
 What is the normality of a 500mL solution that
contains 7g H2SO4?
 MW H2SO4= 98 MW H(1) x2 + MW S (32) + MW O
(16) x 4
 Valence2 H = 2x1 EW = 49g
 N= grams of solute

 EW x volume (L)
 = 7 / 49 x 0.50

 = 0.285 N
SAMPLE PROBLEM # 3 NORMALITY
 To make 500mL of 3 N NaSO4, how much
substance must be weighed?
 MW NaSO4= 142g

 MW Na(23) x 2 +MW S (32) + MW O (16) x4 = 142

 Valence 2 Na= 2 EW = 142/2 = 71

 N= grams of solute

 EW x volume (L)
 Grams of solute =N x EW X volume(L)

 = 3 x 71 x 0.500
 = 106.5 g NaSO4 to make 500 of 3N solution
SAMPLE PROBLEM #4 (NORMALITY)
 In order to make 2 liters of 0.3 N HCl from concentrated
HCl which has a specific gravity of 1.185 and percent
purity of 36.7, what volume of concentrated acid is
required?
 GMW of HCl = 36.5 g MW H (1) + MW Cl(35.5)
 Valence of H =1 EW= 36.5/1=36.5
 N= grams of solute
 EW x volume (L)
 mL HCl= normality x EW x V (L)
 specific gravity x % purity
 = 0.3 x 36.5 x 2
 1.185 x 0.367
 = 50.4 mL
CONVERSION OF % W/V TO NORMALITY
 N= % w/v x10
 EW
 RELATIONSHIP BETWEEN MOLARITY AND
NORMALITY
 NORMALITY = MOLARITY x VALENCE
 MOLARITY = NORMALITY / VALENCE

 NORMALITY IS ALWAYS EQUAL TO OR

GREATER THAN MOLARITY.
 MOLARITY IS ALWAYS EQUAL TO OR
LESS THAN NORMALITY
 MOLALITY
 Amount of solute per 1kg of solvent
 It is expressed as moles per kilogram (mol/kg) or
weight/weight.
 Molecular weight (MW) is obtained by adding the
atomic weight of the given compound.
 FORMULA:

 Molality (m)= grams of solute

 MW x kilogram of solvent
 SAMPLE PROBLEM (MOLALITY)
 A solution contains 15,6 g NaCl dissolved in 500g of water.
Determine the molal concentration.
 GMW of NaCl= 58.5 g MW Na(23) + MW Cl(35.5)

 Molality (m)= grams of solute

 MW x kilogram of solvent
 m = 15.6/ 58.5 x 0.5

 m = 0.53
 MILLIEQUIVALENTS
 The most common way of expressing electrolytes
 A milliequivalent is the equivalent weight
expressed in milligrams.
 Typically, the laboratory is required to convert
milligrams per deciliter (mg/dL) to
milliequivalent per liter (mEq/L)
 FORMULA:

 mEq/L = mg/dL x 10 x valence

 molecular weight (MW)
SAMPLE PROBLEM 1 FOR MEQ/L
 If a solution contains 350mg/dL Na+, how many
mEq/L of Na+ does it contains?
 MW=23

mEq/L = mg/dL x 10 x valence

 molecular weight (MW)
 = 350 x 10 x 1
 23
 = 152 mEq/L
SAMPLE PROBLEM 2 FOR MEQ/L
 A solution containing 12 mg/dL Ca++
 Contains how many mEq/L calcium?

 MW= 40

mEq/L = mg/dL x 10 x valence

 molecular weight (MW)
 = 12 x 10 x 2
 40
 = 6 mEq/L Ca++
 MILLIMOLES
 Millimoles is the molecular weight expressed in
milligrams. (mmol/L)
 Formula:
 mmol/L = mg/dL x 10
 MW
 Example Problem:
 1. Convert a 3mg/dL magnesium to mmol/L
 MW= 24.31
 mmol/L = 3 x10/ 24.31= 1.23 mmol/L
 2. Convert 8.2 mg/dL calcium to mmol/L.
 MW= 40
 mmol/L = 8.2 x 10 /40 = 2.05mmol/L
DILUTION
 In the molar, normal, or percentage solutions, the
amount of solute contained in a given volume of
solution is equal to the product of volume times
the concentration
 Whenever the solution is diluted, the volume is
increased and its concentration decreased, but
the total amount of solute remains unchanged.
 Ratio of the volume of substance to be diluted to
the final volume
 DILUTION = volume of solute

 volume of solution
 DILUTION

 Amount of solute (A) = Volume A x conc,A

 Amount of solute (B) = Volume B x conc.B

 Hence:
 Volume A x conc.A = Volume B x conc.B
 N1 x V1 = N2 x V2
 Sample poblem: Prepare 1Liter of 0.1 N HCl(N2) from
12.1N concentrated HCl (N1) calculate the amount (in mL)
of conc.HCl(V1) to dilute with distilled water (V2)
 N1 V1 = N2 V2
 12.1 x V1 = 0.1 x 1000
 x = 100/12.1 = 8.26 mL
 V1 (x) = 8.26 mL of concentrated HCl to be diluted to 1 liter
distilled water.
 RATIO
 VOLUME OF SOLUTE PER VOLUME OF
SOLVENT

 RATIO = volume of solute

 volume of solvent
 SAMPLE DILUTION
 Calculate the dilution of 0.1 mL serum in
0.9 mL of water
 volume of serum:total volume (serum+water)

 = 0.1 : 10 (0.1 + 0.9)

 dilution is 1:10

 Calculate the dilution of blood when using 50uL

(0.05mL) and 950 uL (0.95mL) of diluting fluid.
 Total volume of body fluid and diluting fluid

 50 + 950 = 1000 uL

 Therefore , dilution of blood: 1000/50 = 20

 The dilution is 1:20

 SAMPLE DILUTION PROBLEM
 Calculate the dilution of urine using 0.5 mL of urine
and 8.5 mL of isotonic saline.
 Total volume of body fluid and diluting fluid
 0.5 + 8.5 = 9mL
 Therefore, dilution of urine : 9.0 / 0.5 = 18
 The dilution is 1 :18
 SERIAL DILUTION:
 Multiple progressive dilutions ranging from more
concentrated solutions to less concentrated solutions.
 Example : A serum was diluted 1:10 then 1:10, then
1:2.What is the final dilution?
 1 x 1 x 1 = 1 final dilution
 10 10 2 200
 MOST COMMON REASONS FOR DILUTIONS
 In the preparation of a working solution from a
stock solution. It is very economical for the
laboratory to purchase concentrated solution and
dilute as necessary to perform the test. The use of
concentrated stock solution, where possible, saces
time, money and storage space.
 If the concentration of the material or solution is
too great to be accurately detremined.
 In the removal of the undesirable
substances(e,g,proteins), solutions are added to
precipitate these substances and dilution has
taken place.
GRADES OF CHEMICAL
REAGENT GRADE OR ANALYTIC
REAGENT (AR) GRADE
 These chemicals are of a high degree of purity and
are used often in the preparation of reagents in the
clinical laboratory for many reagent grade or AR
chemicals; and those that meet their standards are
designed by the letter ACS (American Chemical
Society)
 Important for qualitative and quantitative
analyses; essential for accuracy
 Labels on these reagents either state the actual
impurities for each chemical lot or list the
maximum allowable impurities (percentage of
impurities)
 Uses: trace metal analysis and preparation of
standard solutions.
 CHEMICALLY PURE (CP) GRADE
 These chemicals are sufficiently pure to be
used in many analyses in the clinical
laboratory. However, the designation does
not reveal the limits of impurities that are
tolerated and so, they many not be acceptable
for research and various clinical laboratory
technique unless they have been specifically
analyzed for the desired procedure. It may be
necessary to use this grade when higher
purity biochemicals are not available.
 UNITED STATES PHARMACOPEIA (USP)
AND NATIONAL FORMULARY (NF) GRADE

 These reagents meet the

specifications stated in USP and NF.
They are generally less pure than CP
grade, as the tolerance is specified
such as they are not injurious to
health rather than chemically pure.
PURIFIED, PRACTICAL OF PURE
GRADE

 These chemicals may be used as

starting materials for synthesis of
other chemicals of greater purity but
generally should not be used in the
clinical laboratory.
 TECHNICAL OR COMMERCIAL GRADE
These chemicals are used only
for industrial purposes and are
generally not used in the
preparation of reagents for the
clinical laboratory.
NATIONAL BUREAU OF STANDARDS, COLLEGE OF AMERICAN
PATHOLOGISTS AND THE NATIONAL COMMITTEE FOR
CLINICAL LABORATORY STANDARDS (NCCLS)

 These agencies or bureaus all supply

certified clinical laboratory standards.
 The highest grade or purest chemicals are
available from the National Bureau of
Standards. However, very few such
compounds are available to the clinical
laboratory and they are known as
standards, clinical type.
THREE GRADES OF REAGENT WATER
 Water should be classified in terms of type
instead of the method of preparation,
according to Clinical and Laboratory
Standards Institute(CLSI)
 Filtration is the first step before the three
processes are performed in reagent grade
water preparation.
 Distillation, ion exchange and reverse
osmosis- the processes involved in the
preparation of reagent grade water.
 TYPE 1 REAGENT WATER
 Used for test methods requiring minimum
interference.
 For procedures that require maximum water
impurity for accuracy and precision.
 Should be used immediately (storage is discouraged)
after production.
 For ultramicrochemical analyses, measurements of
nanogram or subnanogram concentrations, tissue or
cell methods(microscopy) and preparation of
standard solutions.
 Uses: flame photometry, AAS, blood gases and pH
enzyme studies. Electrolyte testing, HPLC, trace
metal and iron studies.
 TYPE II REAGENT WATER
 For hematology, microbiology
,immunology and chemistry.
 Acceptable for preparation of reagents
and quality control materials.
 TYPE III REAGENT WATER
 Urinalysis,
parasitology and histology
 For washing glasswares.
 DISTILLED WATER- is the condesate
collected from steam and created when water
is boiled and vaporized. It has been purified
to remove almost all organic materials.
 DEIONIZED WATER- It is prepared by
using deionizer (anion or cation) and it is
free from minerals salts; removed by ion
exchange processes. It has some or all ions
removed but organic materials may be still
present. Is purified from previously treated
water such as prefiltered or distilled water.
 NOTES TO REMEMBER
 Occupational Safety and Health Act (OSHA) requires
manufacturers to clearly indicate the lot number, physical or
bilogical health hazard of the chemical reagents and precautions
for safe use and storage.
 The College of American Pathologists (CAP) recommends that a
laboratory document culture growth, pH and specific water
resistance on reagent grade water.
 Test for water purity- microbiological content, pH, resistivity,
chemical oxygen demand(COD), ammonia, ions and metals.
 Water may bae distilled more than once and each distillation
cycle removes more impurities.
 Water can also be purified by ultrafiltration, UV light,
sterilization or ozone treatment.
 Detergent-contaminated water will have an alkaline Ph.
 Hard water contains calcium, iron and other dissolved elements.
 NCCLS is now Clinical and Laboratory Standard Institute.
MEASUREMENT OF
VOLUME
GLASSWARES
 GLASSWARES
 Clinical laboratory glasswares can be
divided into 5 general types.
1. High thermal resistant glass
2. High silica glass
3. Glass with high resistance to alkali
4. Standard flint glass
5. Low actinic glass
 1. HIGH THERMAL RESISTANT GLASS
 Borosilicate glassware is essentially a sodium-
aluminum borosilicate with an excess of silica
characterized by high degrees of thermal resistance.
Commercial brands are known as kimax and Pyrex. It
has a low alkali content and is free from magnesia-
lime- zinc group of elements. Concentrated alkaline
solutions should not be stored in this glass which will
etch or dissolve the glass and destroy the calibration.
Borosilicate glassware with heavy walls should not be
heated with a direct flame or hot plate nor should one
heat any glass above its strain point, which for Pyrex
is 515 C. if this occurs and the glass is cooled too
quickly.
HIGH THERMAL RESISTANT GLASS

 Strainswill develop and the glass cracks

easily when heated again. Vycor brand is
recommended for use in application involving
high temperature, drastic heat shock, and
extreme chemical treatment with acids and
dilute alkalies. Vycor ware is used primarily
in ashing and ignition techniques. It can be
heated to 900C and can withstand
downshocks from 900 C to ice water.
 2. HIGH SILICA GLASS (OVER 96%)
 This is comparable to fused quarts in its thermal
endurance chemical stability and electrical
characteristics. It is radiation resistant and has good
optical qualities and temperature capabilities. It is used
for precision analytical work and can also be used for
optical reflectors and mirrors.
 Corex brand glassware is a special alumina-silicate
glass strengthtened chemically rather than thermally
which is at least 6 times stronger than borosilicate
glass. It is also better to clouding and scratching.
 Made by removing all elements from borosilicate
3. GLASS WITH HIGH RESISTANCE TO ALKALI

 This is used to handle strongly alkaline

solutions. However, it has only about half
the thermal block or rather shock
resistance of Pyrex glassware and
therefore must be heated and cooled
more carefully.
 Often referred to as “soft glass as its
thermal resistance is much less than of
borosilicate glass.
 4. STANDARD FLINT GLASS

 It is a glass of high thermal with a red color added as

an integral part of the glass. The density of the red
color is adjusted to permit adequate visibility of the
contents, yet give maximum protection tom light-
sensitive materials that causes deterioration. It is used
for bilirubin, carotene and Vitamin A analysis-
photosensitive.
 Used for the manufacture of weighing bottles because
it develops less static surface changes.
 Composed of a mixture of the oxides of silicon, calcium
and sodium.
 5. LOW ACTINIC GLASS
 Thisis soda-lime glass composed of a
mixture of the oxides of silicon, calcium
and sodium. It is lowest in cost and is
readily fabricated in a wide variety of
shapes. It has poor resistance to high
temperature and sudden changes of
temperature and its resistance to attack by
chemicals is only fair. Soda lime pipets
may release alkaline into the pipetted
liquid and cause considerable errors in
certain critical assay procedure.
 MEASURING VESSELS
 1. GRADUATED CYLINDER- commonly called
graduates and are used where less accurate
measurements are required.
 2. BURETS- are long cylindrical graduated
pipettes with stopcock(glass for acid and
rubber for alkali). This is generally used for
titration purposes only.
 3. VOLUMETRIC FLASKS- are frequently
used for the preparation of standard solutions
(solution of known concentration) and for
measuring liquid volume accurately.
 MEASURING VESSELS
 4. PIPETS- there are many kinds of pipets available
for the use in clinical chemistry laboratory each
intended to serve specific function.
 CLASSIFICATION OF PIPETS:

 A. According to manner of calibration(design)

 1. To deliver (TD)- this type of calibration is made

by weighing the necessary volume of water which
when allowed to flow by gravity will deliver the
exact volume. The small amount left in the tip
should not be blow out. This is constant and has
been compensated for during the calibration of the
pipet. The rate at which the fluid flows down is
important and should not be hastened by blowing.
 Calibration of “to deliver” pipets is usually
performed by measuring the amount of
water delivered by the pipet. This
measurement maybe made by weighing
the water delivered and calculating the
volume from its density (V-W/D). Water is
commonly used as the calibrating medium
because it is readily available and it is
similar in viscosity and speed of drainage
to the dilute solution ordinarily employed
in clinical chemistry.
 TO CONTAIN (TC) PIPETS

 Arecalibrated by introducing exact volume

or weight of mercury(Hg), a non-wetting
liquid, equal to volume desired. The pipet
contains the necessary volume, howerer, it
does not deliver the exact volume due to the
tendency of the fluids cling to glass
surfaces. The exact volume can be delivered
by repeated filling, then emptying with the
diluent.
 ACCORDING TO DISPENSING
1. TO BLOW-OUT PIPETTE-
 -same as TD pipette but drops remaining at
tip after delivery to blown out to receiving
vessel.
 - an etched ring/ frosted edge is seen near the
mouthpiece.
 self pipette

 -the user allows the contents of the

 Pipette to drain by gravity

 TYPE OF PIPETTE ACCORDING TO USE
 Volumetric or transfer pipet
 Ostwald-folin

 Graduated or measuring pipette

 Serologic pipettes

 Mohr pipettes

 Micropipettes

 Pasteur pipette

 Automatic Pipettes
 GLASSWARES

Beakers
Graduated measuring cylinders
Volumetric flasks
 EQUIPMENTS USED FOR
MEASURING MASS

Analytical balance
Centrifuge
Upper meniscus

Lower meniscus
 CHAPTER 2
SPECIMEN
COLLECTION
AND OTHER PRE-
ANALYTICAL
VARIABLES
The correct collection of
specimen is essential for
reliable test results. The
laboratory results are only as
good as the specimens received
for testing. It is also the first
step in clinical chemistry
analysis. Poor specimens mean
poor laboratory testing. Proper
technique and procedures are
important to ensure quality
specimens.
 PATIENT IDENTIFICATION

 “PROPER PATIENT
IDENTIFICATION is the first step
in sample collection.” – this is the
prime factor in order to attain
accurate results in the clinical
laboratory. Likewise, proper
techniques in specimen collection
must be strictly followed including
the observance on the
confidentiality of results.
 PATIENT IDENTIFICATION
PROCEDURES:
1. CONSCIOUS
INPATIENT/HOSPITALIZED
PATIENTS
Verbally ask their full name.
Verify the name using the
identification bracelet which includes
first and last names, hospital
units/number, room/bed number and
physician’s name.
PATIENT IDENTIFICATION
SLEEPING PATIENTS
 They are identify in the same
manner as the conscious inpatient.
They must be awakened before
blood collection.
UNCONSCIOUS PATIENTS
 They are identified by asking the
attending nurse or relative;
identification of bracelet.
INFANTS AND CHILDREN
 A nurse or relative may identify the
patient, or by means of an
identification bracelet.
OUTPATIENT/AMBULATORY
PATIENT
Verbally ask their full names,
address or birthdate and
countercheck with driver’s license
or ID card with photo.
If the patient has identification card
or bracelet, same manner as with
hospitalized patients.
 COLLECTION OF SPECIMEN

 TYPES OF SPECIMENS
ANALYZED IN CLINICAL
CHEMISTRY LABORATORY
 Blood
 Urine
 CSF (cerebrospinal fluid)
 Other specimens like ascitic fluid,
peritoneal fluid, etc
 COLLECTION OF SPECIMEN
 TIMING OF COLLECTION:
 Fasting – npo (non per orem); “nothing by mouth”
 generally 8-14 hours

 Glucose- at least 8 hours fasting

 triglycerides- 10-12 hours fasting

 Results of overfasting

 After 48 hours-increase serum bilirubin

 After 72 hours- decrease plasma glucose levels,

increase in plasma triglycerides, glycerol and free

fatty acids with no significant change in cholesterol
 Random – collected anytime
 24-Hour – collected in 24 hours
 Postprandial – collected after meals
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS
 Exercise
 Transient or immediate effects (within the
hour)
 Elevated lactate

 Elevated alanine

 Elevated fatty acids

 Long lasting effects
 Increased activity of muscle enzymes

 AST (SGOT)
 CPK (CK)
 LDH
 ALD (Isoenzyme A)
 Long term effects
 Elevated concentration of sex
hormones
 Testosterone
 Luteinizing hormone
 Sex hormone binding globulin
 Elevated concentration of
steroids
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS
 Fasting
 Generally 8-14 hours
 Elevated blood glucose, potassium, and
lipids like cholesterol and neutral fats are
seen in patients not under NPO and the
reverse is true with inorganic
phosphorous
 Prolonged fasting has been associated
with elevations in serum bilirubin, plasma
triglycerides, glycerol, free fatty acids and
a decrease in plasma glucose.
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS
 Diet
 2-4 hours after fatty meal – increases ALP activity
 High protein diet – increases urea, ammonia and

urates with no significant increase in creatinine

 Serotonin – increase excretion in the urine of 5-

HIAA
 Caffeine – increases concentration of plasma

NEFA
 Causes the release of cathecolamines from the
adrenal medulla and brain tissues
 Increase turbidity or lactescence – triglycerides level
exceeds 4.6 mmol/L (4.0 g/L)
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS

 Posture or position
 Preferably supine (lying) position or
upright sitting position
 Changes in position result to efflux of
filterable substances from the
intravascular space to the interstitial
fluid spaces
 Non-filterable substances increase in
concentration
FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS

 Tourniquet application
 One-minute application
 Prolonged application results
to:
 Venous stasis

 Anaerobiasis
FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS
 Tobacco smoke
 Acute effects
 Increase in plasma NEFA concentration
 Increase in plasma cathecolamines and
serum cortisol (due to nicotine) – affects the
leukocyte peripheral count
 Increase in neutrophils
 Increase in monocytes
 Increase in eosinophils
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS

 Tobacco smoke
 Chronic effects
 Increase in total WBC count

 Increase in blood hemoglobin

values (carboxyhemoglobin)
 Increase in mean corpuscular
Volume (MCV)
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS

 Alcohol ingestion
 Increases plasma
concentration of lactate,
urates, acetate and
acetaldehyde
 Increases gamma glutamyl
transferase concentration and
mean erythrocyte volume
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS

 Stress (anxiety)
 Affects hormone secretion
 Results in hyperventilation
leading to a disturbance in acid-
base balance in the blood
 Increases serum lactate
 Increases plasma free fatty acids
 FACTORS CONTRIBUTING TO THE
VARIATION OF RESULTS
 Drugs
 e.g. Eryrhtromycin, Lincomycin
 Normally affects the liver by
inducing hepatic microsomal
enzymes and therefore could
interfere with determination of
liver function tests.
The correct collection of specimen
is essential for reliable test
results. The laboratory results are
only as good as the specimens
received for testing. Poor
specimens mean poor laboratory
testing. Proper technique and
procedures are important to
ensure quality specimens.
 BLOOD COLLECTION

 Blood is the fluid that the heart

circulates through the body’s arteries,
capillaries and veins. Blood specimens
obtained by phlebotomy.
 Phlebotomy is defined as the puncture
of a blood vessel to collect blood. Blood
for anlysis maybe obtained froms veins,
arteries or capillaries.
 Phlebotomist is a person who performed
or collects blood.
THREE METHODS OF BLOOD COLLECTION

1.skin puncture
2. venipucture
3. arterial puncture
 GENERAL METHODS OF BLOOD SAMPLE
COLLECTION

 CAPILLARY PUNCTURE/SKIN
PUNCTURE/PRICK METHOD
 For micromethod, ultramicromethod and
nanoliter method
 Sample collected is a mixture of blood
coming from the arterioles, venules and
capillaries
 A sharp lancet is used to pierce the skin and
a capillary tube is used for sample collection.
 The indications or method of choice for
Pediatric infant

Geriatrics

Adults who are in extreme obesity,

severe burns and with thrombotic
tendencies
 CAPILLARY PUNCTURE/SKIN
PUNCTURE/PRICK METHOD

Sitesof puncture:
 Palmar surfaces of fingertips
 Plantar heel surface in infants
 Plantar surface of the big toe
 Earlobes
CAPILLARY PUNCTURE/SKIN
PUNCTURE/PRICK METHOD
 Sitesto be avoided:
 Edematous area (swollen with fluid)
 Cyanotic areas (purplish blue in color)
 Scarred area
 Traumatized area (black and blue)
 Heavily calloused area
 ARTERIAL PUNCTURE/ANAEROBIC

 Generally used for the determination of

blood oxygen, carbon dioxide tension and
blood pH (Blood Gas Analysis).
 Blood collected is called arterial blood or
oxygenated blood
 Special training is required for this
procedure
 Tourniquet is not required
 After removing the needle, apply
moderate pressure with 2 x 2 sterile gauze
until bleeding ceases
 Insert needle (still attached to syringe)
in stopper to prevent air from entering
needle
 More difficult to perform because of
inherent arterial pressure, difficulty in
stopping the bleeding and undesirable
development of hematoma.
ARTERIAL PUNCTURE/ANAEROBIC

Sites of puncture:
 Radial artery at the wrist– most
preferred site
 Femoral artery in the groin(fem
tap)
 Brachial artery in the elbow
 Scalp artery
 Umbilical artery
 VENIPUNCTURE

For macromethod
Considered to be the most
commonly used method of blood
collection in Clinical Chemistry
Sample obtained is called
venous blood
 VENIPUNCTURE STEPS
1. PREPARE PAPERWORKS-Carefully look over
requisition slips. Note any specials instruction.
2. IDENTIFY THE PATIENT-a crucial step.
Always ask patient to state his or her full name.
3. VERIFY DIET RESTRICTIONS-a fasting or
non-fasting specimen. Note any medications
that may interfere with testing.
4. ASSEMBLE EQUIPMENT

5. REAASURE AND POSITION THE PATIENT-

Never tell the patient itwill not hurt. The
patient should be seated in a blood drawing
chair or lying down.
 VENIPUNCTURE STEPS
6. APPLY TORNIQUET-the torniquet is applied 3 to 4
inches above the intended site and should never be left in
place for longer than 1 minute.
7. SELECT THE VENIPUNCTURE SITE-have the patient
make a fist; this makes the veins more prominent. A vein
has a bounce or resilience. You must feel the vein.
8. CLEANSE THE SITE- using 70% alcohol, make a circular
motion starting from the center and moving outward.
Allow the site to dry. Do not touch the site after cleaning.
9.UNCOVER THE NEEDLE AND INSPECT- Remove the
cover of the needle and visually inspect the needle or
barbs.
10. PERFORM THE VENIPUCTURE-Anchor the vein using
your nondominant hand and use the thumb to pull the
skin but not tight. Insert the needle into the skin with the
bevel up to 15 to 30 degrees angle with a quick smooth
motion.
 VENIPUNCTURE STEPS
 11. FILL TUBES: order of draw. Always fill tubes
according to the order of draw. If the tube has an additive,
mix well by inverting several times. Tubes will stop filling
when the vacuum is exhausted.
 12. RELEASE TORNIQUET- the torniquet should be
released after the first tube is full. It should not to be left
on for more than 1 minute.
 13. WITHDRAWAL AND NEEDLE DISPOSAL- After the
last tube is filled, release it from the holder, remove the
needle in one swift motion, and immediately apply pressure
to the site. It is not acceptable to have the patient bend
their arm up. Dispose needles in proper container.
 14.LABEL TUBES-all tubes should be labeled with the
patient’s full name, the time and the date. Never label
tubes prior to venipuncture.
 SITES OF PUNCTURE:
 Veins in the antecubital fossa
 Median cephalic vein – most often used for
venipuncture because it is well anchored, has good
blood flow and bruises less easily
 Cephalic vein – rolls and bruises less easily and the
blood flows easily, but tougher to puncture
 Basilic vein – this veins tends to roll easily
 Vein of the longitudinal sinus or sagittal sinus
 Femoral vein
 Wrist vein
 Jugular vein
 Saphenous vein
 Small saphenous vein – at the back of the knee
 Great saphenous vein - front
 Veins on the dorsal portion of the hand
 METHODS OF VENIPUNCTURE
 1.
vacutainer method- multiple sampling
 2. syringe method- single
 Clinicallaboratories perform blood
analyses on venous blood samples
collected by phlebotomy. To collect a
venous blood sample, the phlebotomist
pierces the vein with a hypodermic
needle and draws the blood into a
syringe or uses a commercially
available apparatus specifically
designed for collecting venous blood,
such as the vacuum collection system.
The goal of venipuncture is to obtain a
blood sample from the correct patient into
the correct tube with minimal trauma.
Venipuncture is an invasive procedure and
requires a certain degree of skill.
 VACUUM BLOOD COLLECTION SYSTEM
 The vacuum system consists of a double-
pointed needle, a plastic holder or adapter,
and a series of vacuum tubes with rubber
stoppers of various colors, the colors indicate
the type of additive present. Another kind of
holder is available, which allows resheathing
of the needle with the holder after
venipuncture. Blood collection using the
evacuated tube collection system will produce
the best blood samples for analysis by the
laboratory. The blood goes from the patient
directly into the appropriate test tube.
 BLOOD COLLECTION NEEDLE
 The Vacuum collection needle is pointed at both ends,
with one end shorter than the other. The long end of the
needle is used for insertion into the vein, the shorter end
is used to pierce the rubber stopper of the vacuum tube
and usually is covered by a rubber sheath. The sheath
makes it possible to draw several tubes of blood by
preventing leakage of blood as tubes are changed, this is
called a multi-draw needle. If the short end is not
covered with a rubber sheath, it is a single sample
needle and only one tube of blood can be collected. There
are several sizes of needles available, the size depends
on the length and gauge of the needle that goes into the
vein. Blood collection needle lengths range from 1 to 1 ½
inches. One inch needles are used for routine
venipuncture, 1 ½ inch needles are used for patients
with very deep veins.
 The gauge of a needle is a number that indicates
the diameter of its lumen;
the lumen, also called the bore, is the circular
hollow space inside the needle. The higher the
gauge, the smaller the lumen. The most frequently
used gauges for phlebotomy are 20, 21 and 22.
 The bevel is the slanted opening at the end of the
needle. the phlebotomist performs a
venipuncture so that the bevel of the needle is
facing upward when the needle is inserted into
the vein. Blood collection needles come in single
use, sterile packages, either peel apart envelopes or
plastic cases.
 HOLDER
 The vacuum collection system holder is a
plastic sleeve into which the phlebotomist
screws the double pointed blood collection
needle. Holders are available in two sizes,
one for adult venipuncture and one for
pediatric procedures. All holders are
single use, have an integral safety device
which covers the needle after use and the
entire apparatus is disposed of.
VACUTAINER SET
TYING THE TOURNIQUET
RELEASING THE TOURNIQUET
SELECTING THE VEIN
PALPATING THE VEIN
CLEANSING THE SITE
PERFORMING THE VENIPUNCTURE
COMPLETING THE VENIPUNCTURE- ALWAYS REMOVE
THE TUBE FROM THE BACK OF THE NEEDLE FIRST.
 COMPLICATIONS OF
VENIPUNCTURE
 Immediate Local
Complications:
 Localized
hemoconcentration or
Venous stasis
 Syncope or Fainting
 Failure of blood to enter the
syringe.
 COMPLICATIONS OF
VENIPUNCTURE
 Delayed Local Complications
 Thrombosis of veins
 Thrombophlebitis
 Hematomas
 COMPLICATIONS OF
VENIPUNCTURE

 General Delayed
Complications:
 Serum Hepatitis
 AIDS
COMMON DIFFICULTIES ENCOUNTERED
DURING COLLECTION AND PROCESSING OF
BLOOD

 HEMOLYSIS
 LIPEMIA OR LACTESCENSE
 TYPES OF SPECIMEN FOR CHEMICAL
ANALYSIS
 I .BLOOD
 A. WHOLE BLOOD
 Contains the liquid portion of the blood called plasma and all the cellular
components of the blood.
 1. arterial whole blood- oxygenated blood with a bright red color. It is the
specimen of choice for blood gases and blood pH.
 2. venous whole blood- deoxygenated blood with a dark red color.
Specimen of choice for glucosylated hemoglobin (HbA1c)
B. SERUM
-liquid portion of a clotted blood specimen. Serum proteins include
albumin and globulin, it does not contain fibrinogen since it is consumed
during the clotting process. Serum is the specimen of choice for most
chemical analyses because of the potential interference of anticougulants.
C.PLASMA
- Liquid portion of an unclotted or anticoagulated blood specimen. Plasma
proteins include albumin, globulins and fibrinogen.
 TYPES OF SPECIMEN FOR CHEMICAL ANALYSIS
 II. URINE
TYPES OF URINE SPECIMEN
 A. RANDOM URINE
Collected anytime, a midstream specimen is most desirable for
bacteriologic exam. The first morning sample is generally the most
concentrated and considered the better specimen for evaluation.
 B. 24 hour Urine
 used for assessment of kidney function.
 Collection : Discard the first morning specimen, record the time
and collect every subsequent voiding for the next 24 hours with the
last to be 24 hours after the timing commenced.
 Overcollection occurs if 2 first morning specimens are included in the
collection.
 Preservatives for special tests:
 1. cathecolamines and VMA- bring pH of urine to 1 or 2 with
concentrated HCl.
 2. Porphyrins (stable when alkaline)- add sodium bicarbonate
 3. Adrenal corticosteroids- 2-3 grams boric acid.

-
 TYPES OF SPECIMEN FOR CHEMICAL
ANALYSIS
 III. CEREBRAL SPINAL FLUID

 Examined in the laboratory to established

diagnosis of infection, malignancy or
hemorrahages. Normally obtained through
puncture (“spinal tap”) of the lumbar region(
between L4 –L5) by a physician. 3 aliquots are
collected with name, date and sequential tube
collection number.
 Tube 1. – for chemical and immunologic studies

 Tube 2 – for microbiologic studies

 Tube 3 – for cell count and differential count.

 HANDLING OF SPECIMENS FOR TESTING
 PLASMA OR SERUM PROCESSING
 Plasma or serum should be separated from
cells as soon as possible. Allow the blood to
clot to obtain serum (about 20mins at room
temperature). Centrifuge in original tube
(parent tube) with stopper in place to
prevent evaporation. Draw off into a clean
container labeled with name, date and time.
Put a Stopper , refrigerate or freeze as
appropriate if more than 4 hours will elapse
before analysis.
 UNACCEPTABLE BLOOD SPECIMENS
 1. LIPEMIC (milky appearance) – a cloudy turbid
appearance, presence of lipids, indicate a non-fasting
specimen. Interferes with colorimetric analysis in
chemistry. Plasma or serum containing triglycerides >
400mg/dL or 4.6 mmol/L
 2. HEMOLYZED- Destruction of red cells results in
plasma or serum appearing red to pink. This is due to
release of hemoglobin. Affects potassium and enzyme
testing. Caused by overcentrifugation, excessive
shaking of the sample, traumatic phlebotomy or
intravascular disease. Plasma or serum containing
hemoglobin >200mg/dL
 3.ICTERIC- specimen with a yellowish appearance
because of increased bilirubin content. Plasma or serum
containing bilirubin >25mg/dL or 430umol/L
 CAUSES FOR SPECIMEN REJECTION
1. Unlabeled or improperly labeled tubes
2. Hemolyzed or lipemic specimens
3. Discrepancies between requisition form (lab request
form) and labeled tubes
4. Clots in an anticoagulated tube
5. Wrong tube drawn for a particular test
6. Insufficient amount of blood in the collection tube
(QNS-quantity not sufficient)
7. Poor specimen handling (exposed to light)
8. Specimen contamination
 ANTICOAGULANTS
 Chemical agents that prevent the coagulation
or clotting of blood are anticoagulants.
Anticoagulants are used if whole blood or
plasma is desired.
 Anticoagulants prevent coagulation by:

 Removing the Ca from the blood by

precipitation or binding in unionized form
(e.g. Tri Sodium citrate and EDTA)
 Neutralization of thrombin (e.g. heparin)
 Precipitation of Ca as insoluble salt (e.g.
oxalates)
 OXALATES
 Na, K, NH4 and lithium or di-
oxalate cannot be used for BUN
and electrolyte determinations:
 Lithium oxalate- more soluble
than Na or K
 prevents the formation of a
white precipitate in uric acid
determination.
 Preparation- 1.0 ml of 1%
solution or 10 mg/10 ml of
blood (1 mg/ml of blood)
 OXALATES
Na and K oxalate- concentration of
3 mg or more/ml of blood alters the
electrolyte distribution in blood
interferes with precipitation of
protein (Folin-Wu); gives too low
sugar values and may cause
shrinkage of cells.
Preparation- 0.1 ml 20% solution or
20 mg/10 ml of blood (2 mg/ml of
blood)
 OXALATES

 Double oxalate- Mixed oxalate,

Ammonium- Potassium oxalate, balanced
oxalate, winthrobe fluid, Paul and Heller
fluid- for hematologic purposes.
 Consists of 3 parts by weight of ammonium
oxalate to 2 parts potassium oxalate and
causes no change in red cells due to
balance of conflicting forces.
 The ammonium oxalate tends to swell up
the RBC while potassium oxalate acts in
reverse.
 FLUORIDE

 Na fluoride- used as blood preservative

for blood glucose determination and at
the same time acts as a weak
anticoagulant. It inhibits the enzyme
involved in glycolysis. It is commonly
used with thymol and oxalate whenever
blood must be preserved for later analysis
and blood with Na fluoride is preserved
for 24 hours at room temperature.
 Acts on Ca++ to form insoluble calcium
fluoride.
 CITRATE

 Na and K, ACD (Acid Citrate

Dextrose)- an efficient anticoagulant
for blood transfusion since it is non-
toxic and the salt is rapidly utilized
and excreted by the kidney.
 has the disadvantage of causing
marked shifts of volume and acidity
between red blood cells and plasma.
 Preparation- 6 mg/ml of blood
 EDTA

 Ethylene Diamine Tetra Acetic

Acid- (disodium or dipotassium
salt)- acts as chelating agent which
combines with Ca++ which is
essential for clotting mechanism.
 Preserves the cellular constituents
of the blood.
 Should not be used for NPN and
electrolyte determinations.
 EDTA

Disodium salt- solute to about 10%.

It should be neutralized to pH 7.4
before use by the addition of
NaOH.
Potassium salt- same preparation
as disodium salt
Preparation- 10 mg/ml of blood or 2
gtts of 10% solution/ 10ml of blood.
 HEPARIN
 naturally occurring substance found in the
liver and lungs and it is extremely effective
in preventing the coagulation of blood by:
 preventing the production of plasma
thromboplastin
 inhibiting the formation of the thrombin from
prothrombin
 inhibiting the action of thrombin on fibrinogen;
thus, preventing the formation of fibrin from
fibrinogen
 Not an anticoagulant when acting alone. It
acts in conjunction with certain albumin
fraction of plasma.
CHAPTER 3
ANALYTICAL
TECHNIQUES AND
INSTRUMENTATION
• Analytical techniques and instrumentation
provide the foundation for all measurements
made in a clinical chemistry. This includes
optical instruments, chromatography,
electrochemistry, light emission and
scattering techniques and automation.
• I. OPTICAL INSTRUMENTS
• Instruments that measure light energy.
• Includes
a. Spectrophometers
b. Flame emission spectrophometers
c. Atomic absorption spectrophometers
  Light energy, wavelength and
radiant energy spectrum
 Energy is transmitted via
electromagnetic waves that are
characterized by the FREQUENCY
AND WAVELENGTH.
 Electromagnetic waves is measured in
terms of nanometer (nm) between
PEAKS (MAXIMA) or VALLEYS
(MINIMA) of the wave w/c we call
WAVELENGTH.
The number of vibrations of wave
motion per second known as wave
frequency. The lower frequencies
(longest wavelength) are radio
waves, followed by infrared radiation,
visible light waves, ultraviolet
radiation, x-rays and finally gamma
rays w/c have the highest
frequencies (shortest wavelength).
 PRINCIPLE OF COLORIMETRIC
ANALYSIS
1. That many substances are colored in
solution or can be made to produce a colored
derivative in solution.
2. That the intensity in these colored solutions
is related to the amount of substance in the
solution. For example., a solution which
contains a low concentration of a substance
will appear pale in color, whereas one w/c
contains a high concentration will be dark in
colo
 3.That these colored solutions can
absorb light at a given wavelength in
the visible spectrum. The extent to
w/c a solution absorbs light depends
on the intensity of its color. For
example, only a small amount of
light is absorbed by a pale colored
solution, whereas a lot of light is
absorbed by a dark colored solution
 BEER LAMBERT’S LAW OR BEER’S LAW
 States that the concentration of a substance is
directly proportional to the amount of light
absorbed or inversely proportional to the
amount of transmitted light.
 ABSORBANCE = light blocked, absorbed light
or optical density (OD).
 % TRANSMITTANCE= reflected light,
transmitted light or light scattered
 Absorbance is inversely proportional to %
transmittance.
 %T = It/Io x100
 A = 2 – log%T
 BEER’S LAW
 In colorimetric procedures beer’s law can be applied
to find the concentration of a substance in solution.
Using a reference (standard) solution the
concentration of an unknown test solution can be
determined as follows.

 Concentration = absorbance (unknown) x conc. of ref. of test

 absorbance of reference(standard)
SPECTROPHOTOMETER
• Instrument which measures the
transmitted light of a solution
• is an instrument which
measures the amount of light of
a specified wavelength which
passes through a medium
• Uses prisms and gratings to
isolate a narrow range of
wavelength of light.
 2 KINDS OF LIGHT UTILIZED IN
SPECTROPHOMETRY
 1.VISIBLE SPECTRUM- falls between 400-
700nm
 2. INVISIBLE SPECTRUM

a. ULTRAVIOLET LIGHT (UV) – less

than 400 nm wavelength
b. INFRARED SPECTRUM (IR)- greater
than 700 nm
MIRROR LIGHT ENTRANCE MONOCHRO- EXIT SAMPLE CELL PHOTO- METER
SOURCE SLIT MATOR SLIT DETECTOR READ
OUT

SCHEMATIC DIAGRAM OF A
SPECTROPHOMETER
 COMPONENTS:
Light source – provides incident
light for the system/radiant energy
 Tungsten iodide lamps – most
common light source for the visible
and near infrared region.
 Silicone carbide- for infrared
region.
 Deuterium and hydrogen lamps –
for UV spectrum
 Mercury lamps - HPLC
 COMPONENTS
 ENTRANCE SLIT – reduces entry of stray
light and prevents scattered light from
entering the monochromator.
 STRAY LIGHT – refers to any wavelength
outside the band transmitted by the
monochromator; it causes absorbance error.
 Stray light limits the maximum absorbance
that a spectrophometer can achieve.
 COMPONENTS
 Monochromator- produces light of specific
wavelength from the light source. It isolates specific
or individual wavelength of light.
 the device that allows for a narrow band of
wavelengths
• KINDS OF MONOCHROMATORS;
• COLORED GLASS FILTERS/INTERFERENCE
FILTERS-based on constructive interference
of waves. Light waves enter one side of the filter
and it is reflected to the second surface. Desired
light will reflect back and forth reinforcing the
others of the same wavelengths, undesired
wavelength will cancel out. Simple, least
expensive, not precise but useful
• PRISMS- wedge-shaped pieces of glass
quartz or sodium chloride. A narrow of light
focused on a prisms is refracted as it enters
the more dense glass. For visible and UV
range.
• DIFFRACTION GRATINGS- bends light
(diffraction) and forms wave fronts. Light
that are in phase reinforce one another,
those that are not cancel out and disappears.
For near UV to near infrared spectrum.
Most commonly used, better resolution than
prism. Made by cutting grooves (parallel
grooves) or slits into an aluminized surface
of a flat piece of crown glass.
 COMPONENTS
 EXIT SLIT – It controls the width of light beam
(band pass) –allows only a narrow fraction of
the spectrum to reach the sample cuvette.
 Spectral purity of the spectrophometer is
reflected by the band pass – the narrower the
band pass, the greater the resolution.
 Accurate absorbance measurement requires a
band pass < 1/5 the natural band pass of the
spectrophometer.
 Band pass- the range of wavelength between
the points at w/c transmittance is one half peak
transmittance
 COMPONENTS
• SAMPLE CELL- also known as
cuvettes or analytical cell. It holds the
solution of w/c the absorption is to be
measured.
• Alumina silica glass- most commonly
used
• Soft glass Cuvettes- for acidic solution
• Borosilicate cuvette – for strong
alkaline solution
• Quartz or Plastic cuvette- for UV
measurements
 COMPONENTS
• PHOTODETECTOR-detects and converts transmitted light
into photoelectric energy.
• PHOTOCELL/BARRIER LAYER CELL/PHOTOVOLTAIC
CELL-simplest detector; least expensive; temperature-
sensitive. It requires in external voltage source but utilized
internal electron transfer for current production- low
internal resistance. It is used in filter photometers with a
wide band pass.
• PHOTOTUBE- it contains cathode and anode enclosed in a
glass case. It has a photosensitive material that gives off
electron when light energy strikes it. It require external
voltage for operation.
• PHOTOMULTIPLIER TUBE (PM)- most common type-
measures visible and UV regions.; excellent sensitivity and
rapid response – detects very low levels of light . It detects
and amplifies radiant energy. It should never be exposed to
room light because it will burn out.
• PHOTODIODE-not as sensitive as PM; excellent linearity,
measures light at a multitude of wavelengths and detects
less amount of light.
 COMPONENTS:
METER OR
READ-OUT DEVICE
 It display output of
the detection system.
 Galvanometer/ammet
er
 A moving needle on a
dial or a digital which
indicates the amount
of light passing
through the sample
1) Wavelength selection
2) Printer button,
3) Concentration factor
adjustment,
4) UV mode selector
(Deuterium lamp),
5) Readout,
6) Sample compartment,
7) Zero control (100% T),
8) Sensitivity switch,
9) ON/OFF switch
 HOW TO USE THE
SPECTROPHOMETER
• Select the appropriate wavelength.
• Set the machine to “zero” absorbance. Using a blank (
water, reagent or sample/serum blank), the electrical
read – out of the instrument is set at 100 % T while light
is passing through the blank.
• Sample containing absorbing molecules to be measured is
placed in the light.
• When light of an appropriate wavelength strikes the
cuvette that contains a colored sample, some of the light
is absorbed by the solution, the rest is transmitted
through the sample to the detector.
• The read – out device displays the transmittance of the
colored sample.
• The difference between the amount of light transmitted
by the blank and that transmitted by the sample is due to
the presence of the compound being measured.
A spectrophotometer is being
considered for purchase by a small
laboratory knowing of following
specifications reflects the spectral
purity of the instrument by its
dark current
first step in preparing a
spectrophotometer for an assay to
adjust wavelength selector
 ATOMIC ABSORPTION
SPECTROPHOTOMETRY

Measures
concentration
through the
detection of
absorbance of
electromagnetic
radiation by
atoms instead of
molecules
NEPHELOMETRY

 Itis the
measurement of
light scattered by
small particles an
at angle to the
beam incident on
the cuvette.
 Nephelometers
measure light
scattered at a
right angle to
the light path
TURBIDIMETRY

It is the measurement of the

light blocked by a suspension of
particulate matter as light
passes through the cuvette.
FACTORS AFFECTING TURBIDIMETRY:

sizeand number of the particles

the depth of the tube
cross-sectional area of each
particle
SCINTILLATION COUNTER

 It
is used to
measure the
disintegration
per minute of
time of a
radioisotope.
 POTENTIOMETRY

is the measurement of

differences in voltage at a
constant current.
POLAROGRAPHY

is the measurement of

differences in current at a
constant voltage. Polarography
is used to measure trace metals,
oxygen, Vitamin C and amino
acids concentration.
COULOMETRY

is the measurement of amount of

electricity (in terms of coulombs)
at a fixed potential.
Fluctuation of the needle on a
coulometric type titrator is most
probably due to DIRTY
ELECTRODES.
AMPEROMETRY

the measurement of the amount

of current that flows when a
constant voltage is applied to the
measuring electrode.
CONDUCTOMERY

the measurement of the current

flow between two non-
polarizable electrodes between
which a known electrical
potential is established
FLUOROMETRY

Photometric measurement of
light emitted by a substance that
has been previously excited by a
source of UV light
 CHROMATOGRAPHY
 Separate different substances in the
unknown substance
 Chromatography is based on the principle of
differential solubility
 The amount of the mixture are separated by
a continuous redistribution between two (2)
phases:
 stationary phase
 mobile phase (also called eluent or carrier
fluid)
BASIS OF SEPARATION

1.Rate of diffusion
2. Solubility of the solute
3. Nature of the solvent
PAPER CHROMATOGRAPHY

Aspot of the substance to be

fractionated is placed on the
paper just above the solvent
level. The organic solvent
(mobile phase) moves up through
the paper by capillary action and
various fractions in the sample
move at different rates.
 GEL CHROMATOGRAPHY
When a mixture of small and large
molecules is allowed to pass over
small particles in a column, the
smaller molecules diffuse into the
gel, whereas larger molecules tend to
pass rapidly in the column and
appear in the eluate first. Gel
filtration chromatography is used to
separate compounds on the basis of
molecular weight and size
ION- EXCHANGE
CHROMATOGRAPHY (IEC)
Substances to be separated are
passed on the ion- exchange
column and depending on the
net charge and pH of the
solution; the substance is
absorbed from solution in the
ion- change resin.
LIQUID- LIQUID (PARTITION)
CHROMATOGRAPHY
A highly polar substance tends
to be more soluble in a highly
polar solvent (water), while the
less polar substance tends to be
more soluble in a less polar
solvent, (organic solvent). Thus
following the “Like dissolves
like” principle.
GAS CHROMATOGRAPHY

Itis capable separating and

measuring nanogram and
picogram amounts of volatile
substances.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
Itfollows the concept of selective
adsorption. It applies 4,000-
10,000-lbs/square inch pressure for
the rapid identification and
separation of high molecular
weight components and many
labile biologic compounds such as
peptides, drugs, hormones,
barbiturates, lipids, steroids and
antibiotics
ELECTROPHORESIS

It refers to the migration or

movement of charged particles
in an electric field. A charged
particle or ion will migrate
towards the anode or cathode
depending on the isoelectric pH
(pI) of the solution under the
influence of an externally
applied electric field.
FACTORS AFFECTING RATE OF
MIGRATION:
 NET ELECTRIC CHARGE
 SIZE AND SHAPE OF THE
MOLECULE
 ELECTRIC FLUID
STRENGTH
 NATURE OF SUPPORTING
MEDIA
COMPONENTS OF ELECTROPHORETIC
SYSTEM

1. Support media
2. Electrophoretic chamber
3. Power supply
LABORATORY AUTOMATION

Continuous flow analyzers

Discrete sampling analyzers
Centrifugal fast analyzers
Thin-Film analyzers
DESIGNS:
 Sequential analyzer – performs only
one test at a time
 Batch analyzer – performs only one
kind of test but multiple specimen
 Parallel analyzer – performs
numerous test but only for a single
specimen
 Random access analyzer – performs
test in any order
 CHAPTER 4
QUALITY CONTROL IN
CLINICAL CHEMISTRY
 QUALITY ASSURANCE AND QUALITY
CONTROL
The terms quality control and quality assurance are used to
refer the control of testing process to ensure that test results
meet their quality requirements. The terms have overlapping
definitions and are frequently used interchangeably
QUALITY ASSURANCE (QA) means the practice
which encompasses all endeavors, procedures, formats and
activities directed towards ensuring that a specified quality or
product is maintained.
Includes the pre-analytical and post-analytical phases
of laboratory testing.
QUALITY CONTROL (QC) concerned with the
analytical phase of quality assurance. Involves the process of
monitoring the characteristics of the analytical processes and
detects analytical errors during analysis.
 To obtain reliable results, it is necessary
to have a method that is both
reproducible and accurate.
 QUALITY CONTROL
is the means used to check the
reproducibility and accuracy of assay
methods. It helps to detect errors of
techniques, faults in the preparation of
reference solution and reagents
deterioration of solutions and incorrect
instruments reading.
QUALITY CONTROL
 system of ensuring precision and accuracy
in the laboratory by using quality control
reagents in every series of measurements
 system of techniques to ensure with a
specified degree of confidence that the
result obtained from each series of analysis
is true and correct
 KINDS OF QUALITY CONTROL
1. INTRALAB (INTERNAL Q.C.)- involves the
analysis of control samples along with patient
specimens and evaluation of the results statistically
to determine the acceptability of the analytical run.
Monitors a test method’s precision and analytical
bias.
2. INTERLAB (EXTERNAL Q.C. )- PROFICIENCY
TESTING PROGRAMS that periodically provide
samples of unknown concentration to participating
laboratories. Performance of each laboratory or
participant on an identical specimen can be
evaluated and compared against the performance of
laboratories with the same or similar method.
 PURPOSE OF QUALITY CONTROL
1. Provide a constant check on the reliability
of test results.
2. Give early warning of a method which is
becoming unreliable.
3. Enable acceptable limits to be set for test
methods from which significant changes in
test results can be determined (confidence
limits).
4. Monitor analytical processes, detect
analytical errors during analysis and
prevent reporting of incorrect values.
 OTHER PURPOSES OF QUALITY CONTROL
1. To check the stability of the
machine
2. To check the quality of reagents
3. To check for technical error if any
was committed by the operator
 QUALITY ASSURANCE AND QUALITY
CONTROL

3 phases of Lab. Testing

 Pre-analytical phase
 Analytical phase
 Post-analytical phase
 MainObjective: ensure
that all results are
 reliable
Accuracy

Precision
IMPLICATIONS OF QUALITY CONTROL
 Sensitivity
 The ability of a method to detect and
measure even the smallest amount of
the particular substance tested for.
 It is also the degree by which significant
deviations can be detected
 Analytical sensitivity – able to measure
minute concentration of the analyte
 Diagnostic sensitivity – the test must
always give a (+) result in the presence
of the disease
 IMPLICATIONS OF QUALITY CONTROL
 Specificity
 The ability of a method to measure only the
component desired without the interference of
some other substances present in the same
sample.
 Analytical specificity – able to measure only
one unknown substance
 Diagnostic specificity – the test must always
give a negative result in the absence of
disease
IMPLICATIONS OF QUALITY CONTROL
 Accuracy
 The ability of a method to determine the exact
value of the substance of interest in the sample.
 It is the closeness or the nearness of a test value
(value obtained) to the original value (pre-
determined value).
 FOR EXAMPLE: if a glucose determination is
carried out on a serum sample and the result is
73mg/dL, but the correct concentration of glucose
in the sample is 95mg/dL, accuracy is lacking.
 To have accuracy, standards closest to the
unknown are used.
 IMPLICATIONS OF QUALITY CONTROL

 Precision and Reproducibility

 The ability of a method to give repeated results on
the same sample that agrees with one another.
 PRECISION- is a measure of reproducibility.
 For example, if a number of glucose determinations
are carried out on a serum sample of glucose
concentration 95mg/dL and the results are 73, 72,
75, 74, and 74 mg/dL, one can say that
reproducibility is good even though accuracy is
lacking.
 To have precision, specimens that are analyzed
together with control materials.
 TOOLS OF QUALITY ASSURANCE AND
QUALITY CONTROL
 Standard solution
 For accuracy
 A solution of known characteristics and of known
value or whose concentration is accurately known
 It is composed of one known constituent only and
used as a basis of reference for the calculation of
the value of the unknown.
 100% pure
 Serve as a reference/calibrating substance for
unknown
 Choose the standard nearest to the unknown
PRIMARY STANDARD
 Highly purified chemicals
which may be weighed out
directly in the preparation
of solutions of selected
known concentration.
 Water used in preparation
must be of highest purity.
 Assay value not less than
99.95
 Standards specified by the
committee on Analytical
Reagents (ACS)
 SECONDARY STANDARD
A prepared solution whose concentration is
determined by an analysis of an aliquot
amount of the solution using primary
standard and acceptable reference method.
 Solutions in which concentration cannot be
determined directly from weight of
solute/volume solution.
 Concentration determined by a comparison
with primary standard.
 TOOLS OF QUALITY ASSURANCE AND
QUALITY CONTROL
 Control solution
 For precision
 A solution (either commercially or non-commercially
prepared) composed of several known constituents which
can be run simultaneously with the test to check the
accuracy of the results.
 Is a solution that contain the same constitiuents as those
being analyzed in the patient sample.
 Derived from human blood
 Pooled serum (1yr)
 Controls must be analyzed along with patient samples,
using the same method and are treated like the test
sample. Two levels of controls must be run , a normal
and a pathologic control. There must be run at least
once per shift.
 TWO TYPES OF CONTROL
1. NORMAL CONTROL - a
control product that contains
a physiologically normal
concentration of a particular
analyte.
2. ABNORMAL
/PATHOLOGIC CONTROL -
a control product which
contains a physiologically
high or low concentration of
a particular analyte.
 TWO KINDS OF QUALITY CONTROL
REAGENTS
 Commercially prepared
 Manufactured by different companies
which may come in the lyophilized
(pulverized, dried or powdered) and the
non-lyophilized (liquid) form
 Types:
 Assayed – values are known and given

 Unassayed – values are known but not

given
 TWO KINDS OF QUALITY CONTROL
REAGENTS
 Non-commercially prepared
 Sources for the preparation of non-
commercially prepared QC reagents
 Left-over sera (pooled sera)
 Qualities:
 Non-lipemic
 Non-hemolyzed
 Non-icteric
 Undyed
 Animal blood (cow)
 Blood bank plasma (expired blood)
 Fasting human donor
TOOLS OF QUALITY ASSURANCE AND
QUALITY CONTROL
 Blank
 Solution without the specimen
 With reagent
 Set the reading to zero
 For accuracy
 TYPES OF BLANKS
a. AIR
b. WATER
c. SERUM
d. REAGENT
 CALCULATION AND USE OF QC
STATISTICS
 QC statistics for each test performed in the
laboratory are calculated from the QC
database collected by regular testing of
control products. The data collected is
specific for each level of control.
Consequently, the statistics and ranges
calculated from this data are also specific
for each level of control and reflect the
behavior of the test at specific
concentrations. The most fundamental
statistics used by the laboratory are the
mean [x] and standard deviation [s].
 STATISTICAL CONCEPTS OF QUALITY
CONTROL

Mean
Standard Deviation
Coefficient of Variation
Variance
 STATISTICAL CONCEPTS OF QUALITY
CONTROL
 MEAN- average of a set of values
 Formula 1: Calculating the Mean
[x]
 Where: [x] =Σxn / n
 Σ = sum
 xn = each value in the data set
 n = the number of values in the data
set
The mean (or average) is the laboratory’s best estimate of
the analyte’s true value for a specific
level of control.
To calculate a mean for a specific level of control, first, add
all the values collected for that control.
Then divide the sum of these values by the total number of
values. For instance, to calculate the
mean for the normal control (Level I) in Example 1, find the
sum of the data {4.0, 4.1, 4.0, 4.2, 4.1, 4.1, 4.2}.
The sum is 28.7 mmol/L. The number of values is 7 (n =
7).
Therefore, the mean for the normal potassium control in
Example 1 from
November 1–7 is 4.1 mmol/L (or 28.7 mmol/L divided by 7).
 STATISTICAL CONCEPTS OF QUALITY
CONTROL
2. STANDARD DEVIATION – measure of precision
-measure of the scatter of values around the mean.
 Standard deviation is a statistic that quantifies how
close numerical values (i.e., QC values) are in relation
to each other. The term precision is often used
interchangeably with standard deviation. Another term,
imprecision, is used to express how far apart numerical
values are from each other. Standard deviation is
calculated for control products from the same data used
to calculate the mean. It provides the laboratory an
estimate of test consistency at specific concentrations.
The repeatability of a test may be consistent (low
standard deviation, low imprecision) or inconsistent
(high standard deviation, high imprecision).
Inconsistent repeatability may be due to the chemistry
involved or to a malfunction. If it is a malfunction, the
laboratory must correct the problem.
It is desirable to get repeated measurements of the same
specimen as close as possible. Good precision is especially
needed for tests that are repeated regularly on the same
patient to track treatment or disease progress. For
example, a diabetic patient in a critical care situation may
have glucose levels run every 2 to 4 hours. In this case, it
is important for the glucose test to be precise because lack
of precision can cause loss of test reliability. If there is a
lot of variability in the test performance (high
imprecision, high standard deviation), the glucose result at
different times may not be true.
Standard deviation may also be used to monitor on-going day-to-
day performance. For instance,
if during the next week of testing, the standard deviation calculated
in the example for the normal
potassium control increases from .08 to 0.16 mmol/L, this indicates
a serious loss of precision. This
instability may be due to a malfunction of the analytical process.
Investigation of the test system is
necessary and the following questions should be asked.
1.Has the reagent or reagent lot changed recently?
2. Has maintenance been performed routinely
and on schedule?
3. Does the potassium electrode require
cleaning or replacement?
4. Are the reagent and sample pipettes
operating correctly?
5. Has the test operator changed recently?
FORMULA 2: CALCULATING A STANDARD
DEVIATION [S] FOR A SET OF QC VALUES
 QUALITY CONTROL CHART
 Constructed using the calculated mean
and the SD result.
 Compares the observed control values
with the control limits and provide a
visual displat that is easy to inspect and
review.
 The concentration or observed value is
plotted on the y-axis versus the time of
observations on the x-axis.
 EXAMPLES OF HISTOGRAMS/QC CHARTS
 Shewhart-Levey Jennings Chart
 Most commonly used
 Also referred as a Levey-Jenning chart, S-L/J
 Also known as “dot chart”
 A simple graphical display in which the observed values
are plotted versus an acceptable range of values, as
indicated in the chart by lines for upper and lower limits,
which are commonly drawn as the mean plus or minus 2
standard deviations.
 This chart shows the expected mean value by a solid line
in the center and indicates control limits or range of
acceptable values by interrupted or dashed lines.
 When an observed value exceeds the limits expected, the
analysis should be stopped and the cause of the problem
must be determined.
 Trends and shifts can be identified.
CREATING A LEVEY-JENNINGS CHART
 Standard deviation is commonly used for preparing
Levey-Jennings (L-J or LJ) charts. The Levey-
Jennings chart is used to graph successive (run-to-run
or day-to-day) quality control values. A chart is
created for each test and level of control. The first step
is to calculate decision limits. These limits are ±1s,
±2s and ±3s from the mean. The mean for the Level I
potassium control in
 Table 1 is 4.1 mmol/L and the standard deviation is
0.1 mmol/L.8 .
 Formula 3 provides examples on how ±1s, ±2s and ±3s
quality control
 FORMULA 3: CALCULATING QUALITY
CONTROL LIMITS
 These ranges are used with the mean to construct the
Levey-Jennings chart as shown in Figure 3.
 ±1s range is 4.0 to 4.2 mmol/L

 4.1 – (0.1)(1) = 4.0

 4.1 + (0.1)(1) = 4.2

 ±2s range is 3.9 to 4.3 mmol/L

 4.1 – (0.1)(2) = 3.9

 4.1 + (0.1)(2) = 4.3

 ±3s range is 3.8 to 4.4 mmol/L

 4.1 – (0.1)(3) = 3.8

 4.1 + (0.1)(3) = 4.4

 USING A LEVEY-JENNINGS CHART
TO EVALUATE RUN QUALITY
 The laboratory needs to document that quality control
materials are assayed and that the quality control
results have been inspected to assure the quality of the
analytical run. This documentation is accomplished by
maintaining a QC Log and using the Levey-Jennings
chart on a regular basis. The QC Log can be
maintained on a computer or on paper. The log should
identify the name of the test, the instrument, units,
the date the test is performed, the initials of the
person performing the test, and the results for each
level of control assayed.
 Optional items for the log include: method and the
assay temperature (usually included for enzymes).
There should be room to write in actions taken to
resolve any situation which is identified as “out-of-
control” or unacceptable and a place for
documentation of supervisory review. Once the QC
results are entered into the QC log, they should be
plotted on the Levey-Jennings chart. When the
results are plotted, an assessment can be made
about the quality of the run. The
technologist/technician performing the test should
look for systematic error and random error.
 ERRORS IN QUALITY CONTROL

 Random
 Systematic
 Clerical
 VARIATION (ERROR)
ANALYTICAL ERRORS MAYBE SEPARATED INTO RANDOM AND
SYSTEMATIC ERRORS.

 RANDOM ERROR- error whose source cannot be

identified. Occurs once, non-repeating errors. Difficult
to predict but easy to spot.
 Due to unpredictable cause

 Example: outliers

 An outlier is a value which is far from the main set

of values
 1 outlier in 20 days – interpreted as IN
CONTROL
 2 or more in 20 days- interpreted as OUT OF
CONTROL
 SYSTEMATIC ERROR
 Systematic error is evidenced by a change in the mean
of the control values. The change in the mean may be
gradual and demonstrated as a trend in control values
or it may be abrupt and demonstrated as a shift in
control values.
 Caused by some factory in the analytical system that
can be affect a series of analyses.
 Repeating errors
 Due to definite cause:
 Aging phenomena – variations due to reagents
 Personal bias – variations cause by operator
 Examples: chipped pipet, contaminated reagents.
 TYPES/INDICATORS OF SYSTEMATIC ERRORS
1. TREND – values for the control that continue to either
increase or decrease over a period of 6 consecutive days by
passing the mean .It indicates usually a deteriorating reagent
or changes in the concentration of standards or equipment
error.
2. SHIFT –It is formed by the control values that distribute
themselves on one side of the mean for a period of six (6)
consecutive days. Often deterioration of standard will cause a
shift. Remedied by preparation of a new standard.
3. OUTLIERS
 Are values which are far from the main set of values due
to wild errors
 Conditions when dealing with outliers:
 1 outlier in 20 days (in control)
 2 or more outlier in 20 days (out of control)
GAUSSIAN CURVE

 Also known as
Gaussian
distribution
curve, normal
distribution
curve and
commonly the
bell-shaped curve
YOU DEN PLOT
A 2-mean chart
drawn at right
angles to one
another with
the one set of
values on one
axis another set
of values on the
other axis.
CUMULATIVE SUM GRAPH
 Plottedwith the
accumulated
differences from
the mean of
individual
values with the
middle value
being zero.
WESTGARD MULTIRULES CHART
WESTGARD RULES

1:3s
1:2s
2:2s
4:1s
10:x
R:4s
 INTERPRETATIONS

1. IN CONTROL – when control values

fall within the confidence limit.
- means that the method is
performing properly.
2. OUT OF CONTROL- when control
values fall outside the confidence
limit. Problems maybe developing.
COEFFICIENT OF VARIATION [CV]

 The Coefficient of Variation [CV] is the ratio of the standard

deviation to the mean and is expressed as a percentage. The CV
allows the technologist to make easier comparisons of the overall
precision. Since standard deviation typically increases as the
concentration of the analyte increases, the CV can be regarded as a
statistical equalizer. If the technologist/technician is comparing
precision for two different methods and uses only standard
deviation, he or she can be easily misled. For example, a
comparison between hexokinase and glucose oxidase (two methods
for assaying glucose) is required. The standard deviation for the
hexokinase method is 4.8 and it is 4.0 for glucose oxidase. If the
comparison only uses standard deviation, it can be incorrectly
assumed that the glucose oxidase method is more precise that
thehexokinase method. If, however, a CV is calculated, it might
show that both methods are equally precise. Assume the mean for
the hexokinase method is 120 and the glucose oxidase mean is 100.
The CV then, for both methods, is 4%. They are equally precise.
 VARIANCE

CHAPTER 5 AND 6
CARBOHYDRATES
AND
LIPIDS
 Major food supply and prime source of
energy for humans
 In plants, they are present as starch in the
plant cells and cellulose in plant
framework.
 In animals, CHO are present as glucose
and glycogen and are important source of
energy for vital bodily functions.
 Metabolism of carbohydrates
 PROCESSES INVOLVED IN CHO
METABOLISM
1. GLYCOGENESIS- synthesis of glycogen
from glucose and other sugars.
2. GLYCOGENOLYSIS- breakdown of glycogen
to form glucose and other intermediate
products.
3. GLYCOLYSIS- conversion of glucose and
other hexoses into lactate and pyruvate.
4. GLUCONEOGENESIS- formation of glucose
from non-carbohydrates sources like amino
acids, lactate acid, glycerol,fats and protein.
HORMONES THAT REGULATE
BLOOD SUGAR LEVEL
1. INSULIN – secreted by the beta cells of the
Islets of Langerhans of the pancreas. It
enhances the entry of glucose into the liver,
muscle and adipose tissue which promotes
storage of energy substrate. The secretion
and release is stimulated by the anterior
pituitary gland. It decreases blood sugar
level and stimulates lipogenesis, glycogen
synthesis and storage and protein storage.
Hence, it is also referred as the ‘ hormone of
abundance.”
HORMONES THAT REGULATE
BLOOD SUGAR LEVEL
2. GLUCAGON – secreted by the alpha cells of the
Islets of Langerhans of the pancreas. It increases
blood sugar level by hepatic glycogenolysis and
gluconeogenesis. It stimulates lipolysis and release
of insulin.
3. SOMATOSTATIN – secreted by the delta cells
of the islets of Langerhans of the pancreas. It
inhibits secretion of insulin and glucagon
(modulates the reciprocal relationship of glucagon
and insulin). It also inhibits the release of growth
hormones by the pituitary gland (Growth Hormone
Inhibitory factor)
 4. GROWTH HORMONE or
SOMATOTROPHIC HORMONE- secreted
by the anterior pituitary and have
antagonistic action to insulin (tends to
increase blood glucose level). It stimulates
lipolysis.
 5. THYROXINE or
TETRAIODOTHYRONINE – produced by
the thyroid gland. It stimulates
glycogenolysis which leads to depleted
glycogen stored in the liver. It also increases
rate of gastric emptying and glucose
absorption from the intestines.
 6.EPINEPHRINE(ADRENALIN) AND
NOREPINEPHRINE (
NORADRENALIN)
CATHECHOLAMINES- released from the
chromaffin cells of the adrenal
medulla(inner region) whose action is to
increase glucose concentration by proacting
both liver and skeletal muscle
glycogenolysis.
7. CORTISOL AND CORTICOSTERIODS
(GLUCOCORTICOIDS)- secreted by the
cells of the zona faciculata and zona
reticularis of the adrenal cortex (outer
region of the adrenal gland). These
hormones are diabetogenic by promoting
degradation and increase hepatic
gluconeogenesis.
 CLINICAL CONDITIONS INVOLVING
GLUCOSE ABSORPTION
1. HYPERINSULINISM- condition wherein there is
increased secretion of insulin hormone.
2. INSULINOMA- a tumor of the pancreas which
results to increase secretion of insulin hormone.
3. HYPOINSULINISM- decrease secretion of insulin
hormone.
4. HYPERGLYCEMIA- a condition associated with
increased blood sugar levels.
5. HYPOGLYCEMIA- decreased glucose level in the
blood.
 DIABETES MELLITUS
a group of diseases in which blood glucose
levels are elevated due to deficiency in
insulin secretion or due to abnormal insulin
action.
 The most common disease entity
associated with CHO metabolism.
 Characterized by an insufficient level of
active insulin resulting to increased blood
sugar level and glucosuria (presence of
glucose in the urine.)
CLASSICAL SIGNS AND SYMPTOMS
OF DM (3P’S)
 POLYPHAGIA – excessive eating
 POLYDIPSIA – excessive drinking
 POLYURIA (DIURESIS) – excessive
urine output
 CRITERIA USED TO DIAGNOSE
DIABETES
 FBS level that is greater than or equal to 126 mg/dL (7.0
mmol/L) on at least 2 occasions
 Two-hour postprandial glucose greater than 140 mg/dL (7.8
mmol/L).
 Symptoms of hyperglycemia which include: polyuria,
polyphagia, polydipsia, unexplained weight loss plus a casual
or RBS level of greater than or equal to 200 mg/dL (11.1
mmol/L)
 A two-hour post load glucose of 200 mg/dL or greater than in
an OGTT.
 Exception to these criteria is the diagnosis of gestational
diabetes, which is a condition that develops in approximately
4% of all pregnancies.
 TYPES OF DIABETES MELLITUS
 Diabetes Mellitus TYPE I
 Formerly known as: Insulin Dependent Diabetes mellitus,
Juvenile Onset Diabetes Mellitus
Brittle Diabetes,
Ketosis- Prone Diabetes
 Result of cellular-mediated autoimmune destruction of Beta-
cell of the pancreas
 Absolute insulin defiency/ total lack of insulin secretion
 Occurs at age below 30 years.
 Needs parenteral administrations of insulin
 Ketosis is prominent: KETONEMIA- excessive formation of
ketone bodies in the blood.
 KETONURIA – excessive excretion of ketones in the urines.
 Patient is usually thin
 Glutamic acid decarboxylase autoantibodies
 Tyrosine phosphatase IA-2 and IA2B autoantibodies
TYPES OF DIABETES MELLITUS
 TYPE 2
 FORMERLY KNOWN AS: NON-INSULIN DEPENDENT DM, ADULT
TYPE/MATURITY ONSET DM
 STABLE DIABETES, KETOSIS-RESISTANT DIABETES, RECEPTOR-
DEFICIENT DM
 Insulin resistance with insulin secretary defect
 Insufficient insulin secretion, receptor deficient
 Uses hypoglycemic agent to regulate blood sugar levels
 Hereditary, occurs at age above 30 years
 Patient usually obese, but there are cases where patient is
thin
 Ketosis is rare.
 TYPES OF DIABETES MELLITUS
 OTHER

 Associated with secondary conditions

 Genetic defects of beta-cell function
 Pancreatic diseases
 Drug or chemical induced of beta cell
dysfunction (dilantin and
pentamidine)
 Insulin receptor abnormalities
 Other genetic syndromes
TYPES OF DIABETES MELLITUS
 GESTATIONAL
 A disorder characterized by impaired ability to
metabolize carbohydrates usually caused by a
deficiency of insulin, metabolic or hormonal
changes, occuring in pregnancy and
disappearing after delivery but in some cases
returning years later.
 Screening should be performed between 24 to
28 weeks of gestation ( I hr Glucose challenge
Test- 50g glucose load
 Glucose intolerance during pregnancy
 Due to metabolic and hormonal changes
 TESTS USED TO DIAGNOSE DIABETES
 Fasting Blood Sugar (FBS)
 The test should be performed after an 8 hour fast

 Two-hour post prandial test (PPBS)

 The test is performed two hours after meal

 Oral Glucose Tolerance Test (OGTT)

 Patient to be tested should ingest at least 150 g of CHO 3 days prior
to testing
 Patient must fast the night before the testing is performed
 The patient is challenged orally with 75 g glucose during the test
 Patient should not eat food, drink tea, coffee or alcohol, vigorously
exercise, or
smoke cigarettes during the test
 Venous blood samples collected in gray-top tubes containing fluoride
anticoagulant
SCREENING TEST FOR GESTATIONAL
DIABETES

 A 50 g oral glucose load is recommended as basis of initial

diagnosis. If the 1 hour postload glucose level is 140 mg/dL (7.8
mmol/L), a complete 75g or 100 g three-hour oral glucose
tolerance test should be performed

 Gestational diabetes is diagnosed if the woman is at or

exceeds two of the following four plasma glucose levels during
the complete OGTT
 fasting – 105 mg/dL
 one hour – 190 mg/dL
 two hours – 165 mg/dL
 three hours – 145 mg/dL
 SAMPLE TEST FOR GLUCOSE
DETERMINATION
 FASTING BLOOD SUGAR (FBS)- a specific screening
test for DM requiring fasting from 6-8 hours and used
to detect elevation of blood sugar (glucose)
 RANDOM BLOOD SUGAR (RBS)- determination of
glucose levels on samples randomly collected.
Specifically indicated during insulin shock and
hyperglycemic ketonic coma(emergency cases)
 POST PRANDIAL BLOOD SUGAR (PPBS)- specimen
are collected to 1 to 5 hours after taking his regula
meal. at least 150grams cho should be taken. Blood
sugar concentration should be 120mg/dl at 2 hours. If
hypoglycemia is suspected, do up to 4 to 5 hours PPBS.
 MONITORING TEST FOR DIABETES
MELLITUS
 Glycosylated Hemoglobin/ Glycoselated Hemoglobin (HBA1c)
 It can provide an indication of glucose control over the last three months or
so. Therefore, it is used to monitor how well the patient has maintained
dietary and medication regimens over the last week or month, particularly
on an out-patient basis.
 Determined once in 3 months
 Uses column chromatography or electrophoresis
 Fructosamine
 It refer to glycoselated albumin and other proteins, which
gives a clear picture of more short-term glucose levels (
serum albumin has a half-life of 2 to 3 weeks.
 Once in 3 weeks
 Glycosylated albumin
 Methods: Column chromatography (affinity type) and
Colorimetric assays)
QUANTITATION OF BLOOD
GLUCOSE
 Folin Wu Method – uses PMA
 NELSON-SOMOGYI METHOD

 Accurate but labor intensive and

difficult to automate
 Cu2+ ->Cu+
 Cu+ reduces AMA to molybdenum
blue
QUANTITATION OF BLOOD
GLUCOSE

 Dubowski Method – o-toluidine method

 Most sensitive method
 Uses acetic acid
 O-toluidine is carcinogenic and
poisonous
Glucose + O-toluidine -> Glycosylamine
(green)
 QUANTITATION OF BLOOD
GLUCOSE
 Enzymatic Method - very specific to a substrate
 Glucose oxidase method – first enzymatic reaction used
Glucose + O2→gluconic acid + H2O2
H2O2 measurement:
a. Trinder’s method - H2O2 + Organic dye → colored rxn.
b. Peroxidase Method
H2O2 →H2O + O2
O2 is measured by:
a. Clark electrode
b. Ortho-dianisidine method – initially colorless but when
exposed to O2 becomes orange-brown
 QUANTITATION OF BLOOD
GLUCOSE

 Disadvantage of Glucose Oxidase

Reaction
 Glucose oxidase can only measure beta
glucose
 2 types of glucose

 Alpha-glucose = 35% cannot be

oxidized
 Beta glucose = 65% the only type which
can be oxidized
 QUANTITATION OF BLOOD
GLUCOSE
 Hexokinase Method
Glucose + ATP → G6PO4 + ADP
Measurement of G6PO4:
G6PO4 → Phosphogluconic acid + H
NAD →NADH (colored product)

 Measures both alpha and beta glucose

because of MUTAROTASE
QUANTITATION OF BLOOD
GLUCOSE

 Autoanalyzer method
 Uses alkaline ferricyanide reagent

 CONVERSION FACTOR: 0.055

 CONVENTIONAL UNIT = mg/dl
 SI (system International) unit- mmol/L
 CU x CF= SI unit
 SI/ CF = CU
LIPIDS
 LIPIDS
Organic substance
that are insoluble in
water and soluble in
organic solvents.
 BIOLOGICAL FUNCTION
OF LIPIDS
As primary source of energy
As energy storage
As structural components of
cell membrane
As important signalling
molecules
CLASSIFICATION :
Fatty acid
Triglycerides
Cholesterol
Phospholipids
Fat-soluble vitamins
 Lipoproteins
Chylomicrons
VLDL
HDL
LDL
 LIPIDS
Types of Lipids
 Simple Lipids
 Compound Lipids
 Derived Lipids
 NATURALLY-
OCCURING
MOLECULES
Fats
Waxes
Sterolsfat-soluble
vitamins
Mono and diglycerides
Phospholipids
A. FATTY ACID
Building blocks orb
simplest form of lipid
Source of energy
Used for synthesis of
triglycerides
Not routinely measured
TYPES OF FATTY ACIDS
Unsaturated fatty acids

Saturated fatty acid

 Apolipoproteins
 Apo-A – major protein component of
HDL
 Apo-B – major protein component of
LDL
 Apo-C – major protein component
VLDL
B. TRIGLYCERIDES
Aka triacylglycerol
Storage form of lipid in man
Provide insulation to vital
organs
Transported in the blood by
chylomicrons and VLDL
Causes turbidity of serum
after meals
 Triglycerides
12 hrs fasting requirement
4-6 hrs after meal → peak TAG
level
Serum/plasma → appears
turbid/cloudy
 Milky: white cream layer
II. TRIGLYCERIDES
 Based on the hydrolysis of
triglycerides and the measurement
of glycerol released by the reaction.

A. ENZYMATIC (Glycerol Kinase)

B. NON – ENZYMATIC METHOD
1. Van – Handel and Silversmith
2. Hantzch Condensation (
fluorometric method)
 Triglyceride Determination
 Zilversmith and Van Handel
 1st step: extract using organic solvents (alcohol
and ether) → Bloor’s reagent
 2nd step: add KOH → for the hydrolysis of
triglyceride into fatty acids + glycerol (alkaline
hydrolysis)
 3rd step: measurement of glycerol
 Oxidize glycerol by periodate solution →
formaldehyde
 4th step: color reaction of formaldehyde
 Formaldehyde + chromotrophic acid → pink

chromophore
 Triglyceride Determination
 Enzymatic Method
 Uses Lipase – no extraction needed
TAG →F.A. + glycerol
Glycerol kinase →pyruvate kinase
(colored product)
TOTAL CHOLESTEROL
 2 Types:
 Free cholesterol – 30%
 Esterified cholesterol – 70%

 2 Sources
 Exogenous – food (15%)
 Endogenous – hydrolysis of acetyl coenzyme A
Methods of Lipid Determination
 Liebermann-Burchardt (L-B) Procedure
 one step direct method (simplest approach)
 cholesterol + H2SO4 + acetic anhydride = oxidation
products
 measure at 4 10 nm
 Other serum constituents such as hemoglobin and
bilirubin absorb strongly in this region and may
produce falsely elevated values
Methods of Lipid Determination
 Abell-Kendall Method
 Precipitation of cholesterol esters after extractiuon
separate esterified from free cholesterol, permitting
measurement of only the free fraction
 Extraction: specimen + zeolite = cholesterol
 Cholesterol esters – hydrolysis – free cholesterol
DETERMINATION OF HDL
CHOLESTEROL
 Precipitation of VLDL and LDL by the addition
of reagent containing heparin-MgCl2; dextran
sulfate-MgCl2 and phosphotungstate- MgCl2
 Centrifugation – HDL is the only LP remaining
in the supernate
 Determination of cholesterol by enzymatic
method
Color Reactions for cholesterol
 Liebermann-Burchard reaction
 Most popular color reaction
Cholesterol + Acetic anhydride.H2SO4
→cholestadienyl monosulfonic acid (bluish green
compound)
 Salkowski reaction
 Most commonly employed
 Methods for total cholesterol
determination
 Bloor’s method
 1st step: extract using Bloor’s reagent
 2nd step: Liebermann-Burchard
 End color: Bluish green
 Abell-kendall method
 1st step: extract using Zeolite
 2nd step: LB reaction
 Enzymatic method
Free cholesterol [o] →H2O2
2 enzymes involved:
 Cholesterol esterase – converts esterified cholesterol into
free cholesterol
 Cholesterol oxidase – oxidize into H2O2
Low density lipoprotein
Determined using the Friedewald equation:
TC = HDL + LDL + VLDL
Hence:
LDL = TC – (HDL + TG/5) if TG <400
LDL = TC – (HDL + TG x 0.16) if TG >400
LDL = TC – (HDL + TG/2.175) if TG is in mmol/L
PHOSPHOLIPIDS
Most abundant lipid
Important component of the
cell membrane because of its
polar and non-polar section
Not routinely measured
 FORMS :
1. Lecithin ( 70 %)
>aka phosphatidyl
choline
>act as lung surfactant
2. Sphingomyelin ( 20 %)
3. Cephalin (10 %)
LIPOPROTEIN
Lipid -protein complexes
Transport lipids in the blood
Includes HDL, LDL, VLDL
Classified according to
density
> by centrifugation
> by electrophoresis
 DENSITY OF LIPOROTEINS
INCREASES AS:

Protein content
increases
Lipid content decreases
Size become smaller
TYPES :
HDL –High Density
Lipoprotein
LDL – Low Density
Lipoprotein
VLDL – Very Low
density lipoprotein
 HDL
 Aka alpha lipoproteins or good cholesterol
 Produced by the liver and intestine
 Smallest and heaviest lipoprotein
 Transports cholesterol back to the liver for
synthesis of bile acids or VLDL
 High levels associated with decreased risk
of developing atherosclerosis.
 LDL
Aka beta lipoprotein or bad
cholesterol
Formed from the breakdown of
IDL
Transports cholesterol to the
peripheral tissues
High levels are associated with
increased risk of developing
atherosclerosis
VLDL
Aka pre beta lipoprotein
Produced by the liver
Transports liver
synthesized triglyceride to
muscle and adipose cells.
CHYLOMICRONS
Produced by the intestine
Largest and least dense
lipoprotein
Transport ingested triglycerides
to adipose tissue and muscle
cells
Accounts for lipemia after meals
MINOR LIPOPROTEINS
Intermediate
density lipoproteins
Lipoprotein A
 APOLIPOPROTEINS
 Protein and hydrophilic component of
lipoproteins
FUNCTION :
> Structural support for lipoproteins
> Acts as co – factors for lipid
catabolism
 Five major apolipoproteins (Apo A, B,
C, D and E)
 Most requested test is Apo A and B
APO A
Found primarily in HDL

MEMBERS :
a. Apo A-I

b. Apo A-II
APO B
Major protein component of
LDL

MEMBERS :
a. Apo B – 100

b. Apo B - 48
ENZYMES
Lipoproteins Lipase – hydrolyzes
triglycerides from lipoproteins
LCAT (Lecithin, Cholesterol
acyltransferase) – forms
cholesterol esters for storage
HMG CoA reductase – catalyzes
production of cholesterol.
 LIPIDS AND LIPOPROTEIN
ANALYSES

ESTIMATION OF
PLASMA LIPIDS
Cholesteroland
Triglycerides are the
plasma lipids of interest
in the diagnosis and
management of
lipoprotein disorders.
 I. CHOLESTEROL
MEASUREMENT

Enzymatic method
(cholesterol oxidase)

Non– enzymatic
method
NON- ENZYMATIC METHOD
 Liebermann –burchard reaction (L-B
reaction)
>color reaction for cholesterol
METHOD :
1. Abell-Kendall
>Three method
a. cholesterol esters are hydrolyzed with
alcoholic KOH
b. unesterified cholesterol is extracted with
petroleum ether
c. measures using Liebermann-Burchard
reaction
 CHOLESTEROL NORMAL
VALUE

Conventional : 150 – 250 mg/dL

SI Units : 3.88 – 6.47 mmol/L

Conversion factor : 0.026

 TRIGLYCERIDES NORMAL
VALUE

Conventional : 10 – 190 mg/dL

SI units : 0.11 – 2.15 mmol/L

Conversion Factor : 0.0113

 ESTIMATION OF
LIPOPROTEINS

Ultracentrifugation
Electrophoresis
Polyanion
precipitation
 DETERMINATION OF HDL
CHOLESTEROL
Precipitating reagents such
as divalent cations and
polyanions are used to
remove all lipoproteins
except HDL.
Enzymatic method for total
cholesterol is used to
quantitate HDL.
 DETERMINATION OF LDL
CHOLESTEROL
 Friedewald

VLDL= trigly = mg/dL

5

VLDL = trigly = mmol/L

2.175
DE LONG

VLDL= trigly = mg/dL

6.5

VLDL = trigly = mmol/L

2.825
CLINICAL CORRELATIONS
 Atherosclerosis
 Myocardial infarction

 Hypertension

 Hyperlipoproteinemia

 Hipolipoproteinemia

a. Abetalipoproteinemia
(Bassen-Kornzweig Syndrome)
b. Tangier’s disease
 DETERMINATION OF
CHYLOMICRONS

 Standing plasma test

>chylomicrons, if present in appreciable
quantitie,`are detected using the
standing plasma test.
>an aliquot of plasma is placed in a test
tube and allowed to stand in the
refrigerator at 4ºC undisturbed
overnight
>chylomicrons accumulate as a floating
cream layer and can be detected visually.
 CHAPTER 7

PROTEIN
 PROTEIN
 These are high molecular weight
(macromolecules) organic compounds
composed of amino acids that are
combined together by peptide bonds
(amide linkages) to form polypeptide
bonds.
 MOLECULAR SIZE

biologically active proteins are

macromolecules range in
molecular weight from 6000 for
insulin to several million for some
structural proteins.
STRUCTURE

backbone of each protein molecule is a

continuous chain of carbon and nitrogen
atoms joined through peptide bonds
between adjacent amino acids. Wherein a
free amino group is located on one end (N-
terminal end) and a free carboxyl group on
the other (c-terminal)
PROTEIN STRUCTURES
 PRIMARY STRUCTURE- Presents as a linear sequence of
amino acids.
 SECONDARY STRUCTURE- twisted shape of primary
structure due to rotation of bonds. Shape maybe alpha helix
, pleated sheet, or random coil.
 TERTIARY STRUCTURE- three-dimensional structure
that forms when the amino acid chain folds on its self.
 QUARTERNARY STRUCTURE- individual proteins or
monomeric subunits that form more stable complexes as
dimers, trimmers, etc.
CHARGE AND ISOLELECTRIC POINT

 The pH at which particular protein has a net

charge equal to zero is called its isoelectric point
(pl).
 Amino acids assume a double charge. Amino
group is positively charged and the carboxyl
group is negatively charged, resulting to a net
charge of zero.
 Because of their amino acid composition, proteins
can bear a positive or negative charge; hence they
called amphoteric substances. Because of this,
they can act both as weak bases and weak acids,
thus explains the buffering action of proteins.
 IMMUNOGENICITY
Most of the plasma proteins are
synthesized in the liver and secreted
by the hepatocyte into the circulation.
The immunoglobulins are exceptions
because they are synthesized by
plasma cells.
 DISTRIBUTIONS, CATABOLISM, AND
NITROGEN BALANCE

1.Proteins are distributed between two major

compartments, the extravascular and the
intravascular compartments.
2.During catabolism, proteins are hydrolysed to
their constituents amino acids.
 Amino acids are determined, producing
ammonia and ketoacids
 Ammonia is converted to urea by the
hepatocytes and is excreted in the urine.
 Ketoacids are oxidized by means of Kreb’s cycle
and converted to glucose or fat.
DISTRIBUTIONS, CATABOLISM, AND
NITROGEN BALANCE
3.Ordinarily, a balance exists between protein
anabolism and catabolism. This is called the nitrogen
balance.
 Negative nitrogen balance: when protein catabolism
exceeds protein anabolism, wherein the nitrogen
excreted exceed that ingested. Occurs where there is
excessive tissue destruction, such as burns, wasting
disease, continual high fevers, or starvation.
 Positive nitrogen balance: when anabolism is greater
than catabolism. Found during growth, pregnancy,
and repair processes.
 Function of Proteins
 Enzymes
 biochemical catalysts
 Structural proteins
 Provide structural support for the body, a tissue or a cell
 Usually long, fibrous molecules
 e.g. collagen – found primarily in the bone
 keratin – hair and nails
 Contractile proteins
 Involve in the contraction and relaxation of muscles
 Usually long, fibrous materials
 Antibodies
 neutralize foreign materials
 Transport proteins
 Serve a vital function in carrying materials from one part of the
body to another through circulation
 Peptide hormones
 Regulate the metabolism
 Function of Proteins
 To act as pH buffer
 To provide factors necessary for normal blood
caogulation (ex. Fibrinogen, prothrombin,
antihemophilic factor)
 To maintain normal osmotic pressure in the
blood.
 To provide necessary enzymes (bichemical
catalysts) in the blood (ex. Portienase,
peptidase, amylase, etc.)
 Protein Profile

Total Serum Protein

Albumin
Globulin

 Used for the evaluation of

synthetic function of the liver
LABORATORY TESTS OF PROTEINS

Protein analysis provide us with

information regarding the nutritional
status and the presence of severe
disease states involving the liver, kidney
and bone marrow. The laboratory
evaluation of plasma proteins is
performed using the following methods:
 GENERAL METHODS
 I. SALT FRACTIONATION
METHODS
 II. TURBIDIMETRIC METHODS
 FLOCCULATION/TURBIDITY
TEST
 III. ELECTROPHORESIS
 IV. ULTRACENTRIFUGATION
 I. SALT FRACTIONATION
 Will differentiate protein according to their solubilities
using different concentration of salt solution.
 Ammonium sulfate- most frequently used salt.
 SOLUBILITY PROPERTIES
 ALBUMIN – soluble in : water-dilute and
moderately concentrated salt sol’n.
INSOLUBLE- hydrocarbon solvents sat’d salt
solution highly conc. salt sol’n.
GLOBULIN- soluble in : hydrocarbon solvents, weak
salt solution.
Insoluble in : water, sat’d salt solution , conc, salt
solution
ALBUMINOIDS- Characterized by being insoluble
in most common regents.
 ELECTROPHORESIS

 provides quick and useful information

regarding altered levels of protein groups
within the circulation.
 ULTRACENTRIFUGATION

 Will differentiate protiens according to their

differences in molecular densities; thus,
heavier molecules settle faster than lighter
molecules.
 TOTAL PROTEIN METHODS:

 KJELDAHL
-acid digestion of protein with measurement of
total nitrogen
-reference method, assume average nitrogen
content of 16%
-involve a 2-step reaction:
KJELDAHL
1. Kjeldahlization – conversion of nitrogen to
ammonia
H2SO4
Nitrogen NH3
TOTAL PROTEIN METHODS:
2. Ammonia measurement

Nessler’s reaction
Nessler’s reagent: double iodide of mercury and potassium
Gum ghatti
Ammonia + Nessler’s rgt yellow solution
(ammonium dimercuric iodide)

 Berthelot’s reaction
Na nitroprusside
Ammonia + alkaline hypochlorite indophenol blue
 TOTAL PROTEIN METHODS:
 BIURET
-formation of violet colored chelate between
cupric ions and peptide bond
-routine method, requires at least 2 peptide
bonds

 Composition of biuret reagent:

1.Cupric ions-breaks the peptide bonds
2.Tartrate salt-keeps copper in solution
3.Potassium iodide-stabilizes cupric ions
TOTAL PROTEIN METHODS:

 REFRACTOMETRY
-measurement of refractive index which
reflects the concentration of proteins
-rapid and simple
TOTAL PROTEIN METHODS:
 ULTRAVIOLET ABSORPTION METHOD
-proteins in solution absorb ultraviolet light at 280 nm
(A 280), mostly to tryptophan, tyrosine, and
phenylalanine.
-molar absorptivity used for accurate conversion of A
280 readings to protein concentration, since different
proteins contain different amounts of three amino
acids.
-A 280 of mixture of proteins is not an accurate
measure of protein content, since molar absorptivities
vary greatly between different proteins.
TOTAL PROTEIN METHODS:

 FRACTIONATION BY ELECTROPHORESIS
-separates proteins on basis of their electric
charge.
PRINCIPLE: with the use of Barbital (Veronal)
buffer, at pH 8.6 all serum proteins become
negatively charged and migrate towards the anode
and produce a pattern of separation on an
electrophoretogram.
 FRACTIONATION BY
ELECTROPHORESIS
 A.Of the solid support media, cellulose acetate and
agarose gel have predominated in the laboratory due
to their ease of use, low cost and commercial
availability.
 B. After migration, stains may be used to locate and
identify the separated fractions on the sample such
as Ponceau S. Bromphenol blue, and Coomassie
brilliant blue.
 C. Fractions are quantitated using densitometer.
 D.Total proteins are separated as 5 distinct bands.
From fastest to slowest: albumin, alpha-1-globulin,
beta globulin, and gamma globulin.
 SUMMARY OF THE MAJOR PLASMA PROTEINS
Plasma Protein Function
Albumin Regulation and maintenance of plasma
volume and distribution of extracellular
fluid.
Also functions as a carrier protein.
Alpha1 Globulin Anticoagulant effect
Alpha1Antitrypsin Lipid transport
HDL

Alpha2 Globulin Copper transport

Ceruloplasmin Binds free haemoglobin
Haptoglobulin Anticoagulant effect
Alpha2 Macroglobulin Lipid transport
VLDL

Beta-Globulin Iron transport

Transferrin Component of complement pathway
C3 and C4 Fibrinolysis
Plasminogen Fibrin formation
Fibrinogen Lipid transport
LDL

Gamma-Globulin Each immunoglobulin has a different

Immunoglobulins antibody function
IgA, IgD, IgE, IgG and IgM
PATTERNS OF PROTEIN ABNORMALITY
-visual inspection of electrophoretogram
provides a more detailed information about
individual proteins because some diseases
have chracteristics patterns
-interpretation of results depends on visual
inspection to identify abnormal patterns or
aberrant bands.
-serum electrophoresis patterns and
clinicopathologic correlations are tabulated in
table.
 SERUM ELECTROPHORESIS
Common SPE pattern Comments

PATTERNS
Normal SPE pattern (normal
pattern is dotted)
Used to compare with the
patient’s pattern to detect
changes in peak slope or
shape
Monoclonal gammopathy
Marked, single spike in
SERUM ELECTROPHORESIS
the gamma region.
PATTERNS
Caused by multiple
myeloma, heavy chain
diusease or
Waldenstrom’s
macroglobulinemia.
SERUM ELECTROPHORESIS
Nephrotic syndrome Decreased in most serum
proteins
PATTERNS Increased A2 fraction
Nephrotic syndrome generally
results in a loss of most proteins
except alpha2 macroglobulins
because it is a very large protein.
Cirrhosis Increased IgA
SERUM ELECTROPHORESIS
“beta-gamma bridging
PATTERNS or infusion of the beta
and gamma regions is
very characteristic for
cirrhosis. Pattern
suggests increased IgA
which migrates in the
valley between beta and
gamma.
 ALTERATIONS IN SERUM TOTAL
PROTEIN
HYPERPROTEINEMIA HYPOPROTEINEMIA

Dehydration
Monoclonal or polyclonal Protein loss
gammapathies - Nephrotic syndrome, blood
loss, burns
Protein catabolism
- Inflammation, malignancy
Protein synthesis
- Liver disease
- Amino acid intake
 Determination of Total Serum Protein
 Conversion Factor: 10
 Micro-Kjehldahl-Nessler

 Biuret Method – uses alkaline copper

tartrate rgt to cut peptide bonds; violet end
color
 SPE
 Serum Protein Electrophoresis
 Proteins @ low pH: positively charged
 Proteins @ high pH: negatively charged
 pH of SPE: 8.6
 Polyacrylamide gel
 Rate of migration:
 Molecular weight
 pH
 # of charges
 Color reagent/spray dyes:
 Ponceau S – Red
 Bromphenol Blue
 Amido Black
 Coomasie Brilliant Blue – for CSF
 Silver Stain
 Output: electrophoregram measured by a
densitometer
DETERMINATION OF INDIVIDUAL
OPROTEINS
1. ALBUMIN
 - protein present in highest concentration in the serum
(2/3 of total plasma proteins)
 - produced by liver
 FUNCTIONS:
 -regulation of normal osmotic pressure
 -general transport protein which also serves as a mobile
repository of amino acids from the liver where it is
synthesized.
 -general methods for albumin is tabulated in table.
METHOD PRINCIPLE COMMENTS
Salt Globulins are Labor intensive
precipitation precipitated with 22-
GENERAL METHODS FORsulphate,
26% sodium ALBUMIN
albumin in supernatant
is quantitated by Biuret
reaction.
Dye binding Binding increases the Routine method
absorbance of the dye Example of dyes used:
proportionate to the 1. Bromcresol green
concentration dye (BCG)-most
common
2. Bromcresol purple
dye (BCP)-more
precise
3. 2,4-
hydroxyazobenzene
benzoic acid (HABA)
4. Methyl orange

electrophoresis Proteins separate based Accurate, normally the

on electric charge largest protein fraction
(40-60%)
Albumin determination
 Precipitation using Na2SO4; residue (globulin);
filtrate (albumin)
 Analyzed using

 Micro-Kjehldahl Nessler
 Biuret Method
 Dye-Binding Method
 Bromcresol green

 HABA

 Methyl orange
 Albumin-Globulin Ratio
 1.5-3.5/1

 Inverted A/G ratio – seen in multiple

myeloma
ALTERATIONS IN
HYPERALBUMINEMIA SERUM ALBUMIN
HYPOALBUMINEMIA
dehydration Albumin loss
- Nephrotic syndrome, blood
loss, burns
Albumin catabolism
- Inflammation, malignancy
Albumin synthesis
- Liver disease
- Amino acid intake
 DETERMINATION OF INDIVIDUAL
OPROTEINS
2. GLOBULIN
- total globulins are measured by subtracting albumin from the total
protein concentration:

TP – albumin = globulin

Normal values:
Conventional SI unit
Total protein 6.0-7.8 g/dL 6078 g/L
Albumin 3.2-4.5 g/dL 32-45 g/L
Globulin 2.3-3.5 g/dL 23-35 g/L

Conversion factor: 10
DETERMINATION OF INDIVIDUAL
OPROTEINS
3.FIBRINOGEN
- most abundant of coagulation factors responsible for the formation of fibrin
clot
- only protein found in plasma which is not found in serum
FORMULA:
TP (PLASMA) - TP (SERUM) = FIBRINOGEN
METHODS OF DETERMINATION:
Parfentjev method
Fibrinogen is precipitated with ammonium sulphate and sodium chloride
from a citrated plasma. A dilution of mixture is then read
spectrophotometrically at 510 nm.
Howe’s method
Fibrinogen is precipitated with calcium chloride and assayed using the
Kjeldahl method.
NORMAL VALUE: 200-400 mg/dL (2.0-4.0 g/L)
 MISCELLANEOUS PROTEINS
1. Alpha1 antitrypsin
Acute phase reactant, makes up 90% of alpha-1 region
Function neutralize trypsin like enzymes that can cause damage
to structural proteins.
Decrease may cause early onset of emphysema or infatile
hepatitis
2. Alpha1 fetoprotein
Principal fetal protein, no known adult function
Increase concentration in adult may indicate hepatocellular
tumor
3. Haptoglobulin
Binds and transports free hemoglobin
Decreased in intravascular hemolysis
MISCELLANEOUS PROTEINS
4. Ceruloplasmin
Copper transport protein
Decreased in Wilson’s disease; increased in copper toxicity
5. Alpha2 macroglobulin
One of the largest protein in plasma
Dramatic increase in nephrotic syndrome
6. Transferrin
Transports iron
Increased in iron deficiency anemia, decreased in
inflammation
MISCELLANEOUS PROTEINS
7. Bence Jones protein
Abnormal protein in urine of patients with multiple
myeloma
Demonstrated by heart and acetic method:
˜Bence Jones precipitates when solution is heated at 60-
65˚C, becomes soluble when heated at 100˚C and re-precipitates
when the solution cools to 60-65˚C.
8. Cryoglobulins
Serum globulins that precipitate on cooling and re-dissolve
on warming.
Specific Plasma Proteins and Functions
 Prealbumin
 It is also called thyroxine-binding prealbumin (TBPA)
or transthyretin (TTR).
 Albumin
 It is the most abundant protein in the plasma.

 It serves as a mobile repository of amino acids for

incorporation into other proteins.
 It is the general transport protein or carrier.

 Elevations of serum albumin concentration

occur in
 Dehydration

 Prolonged application of tourniquet for

venipuncture
 Specific Plasma Proteins and Functions
 1-antitrypsin
 It the major component of the 1-globulins.
 It has the capacity to combine with trypsin and inactivate it.
 It is one of the serum glycoproteins that rise in response to
acute inflammation.
 2- macroglobulin
 When other lower molecular weight proteins are lost, its

concentration rises 10-fold or more in nephrotic syndrome.

 Haptoglobin
 It combines with hemoglobin released by lysed red cells in

order to preserve body iron and protein stores.

 Transferrin
 It transports ferric ions from the iron stores of intracellular
or mucosal ferritin to bone marrow
 KIDNEY
 Thekidney is primarily concerned in the
elimination of nitrogenous waste products
that one might think of it purely as an
excretory organ. The kidney should not be
viewed only as an organ which removes end-
products of metabolism and other waste
products of foreign substances dissolved in the
plasma but also makes continuing
adjustments in the concentrations of normal
constituents in the plasma-regulatory.
 KIDNEY
 THE GENERAL FUNCTIONS OF THE
KIDNEYS
 ELIMINATION OF METABOLIC WASTE
PRODUCTS THROUGH THE FORMATION
OF URINE
 REGULATION OF THE PLASMA AND
WATER VOLUME.
 REGULATION OF IONIC EQUILIBRIUM
 MAINTENANCE OF ACID BASE BALANCE
 ENDOCRINE FUNCTION
 ELIMINATION OF METABOLIC WASTE PRODUCTS THROUGH
THE FORMATION OF URINE

 The functional unit of the kidneys that is responsible for

these functions is the NEPHRON which approximately 1-
1.5 millions/kidney ( 2,3 millions) in humans. Each consists
of a glomerulus protected by Bowman’s capsule and
nephric tunules.
 The basic function of the nephrons is to clear the blood
plasma of ‘unwanted substances” as it passes thru the
nephrons. The substances which must be cleared include
metabolic end products such as creatinine, urea uric acid
and sulfates. In addition, substances which tend to
accumulate such as the electrolytes are also cleared out by
the nephron.
 The mechanism by which the nephrons clear plasma of
unwanted substances are:
1. It filters a large portion of the plasma thru glomerular
membrane. (Glomerular filtration)
 ELIMINATION OF METABOLIC WASTE PRODUCTS
THROUGH THE FORMATION OF URINE
 2. as this fluid flows thru the tubules, substances valuable to
the body such as water, electrolytes, amino acids and glucose
are reabsorbed back into the plasma (TUBULAR
REABSORPTION)
 3.in addition to reabsorbing of many solutes from the tubular
fluid, the tubules also remove certain substances from the
peritubular blood to be included in the peritubular fluid
(TUBULAR SECRETION)
 Thus , three separate processes: GLOMERULAR
FILTRATION, TUBULAR REABSOPTION (removal of water
and solutes from tubular fluid) and TUBULAR SECRETION(
secretion of solutes into tubular fluid) are involved in the
process of urine formation
 REGULATION OF THE PLASMA AND WATER
VOLUME.
Sixty percent (60%) of an average man is water and
20% of this is in the extracellular fluid (ECF)
compartment. Maintenance of body fluid volume is the
prime function of the kidneys due to its ability of
concentrating and diluting urine.
• REGULATION OF IONIC EQUILIBRIUM
• Electrolytes balance is important in homeostasis. The
kidneys prominently affects ionic balance thru its
reabsorptive and secretory capacity. Concentration of
sodium, potassium, bicarbonate, calcium as well as
other cations and anions are regulated in the kidneys
depending on body’s needs.
 MAINTENANCE OF ACID BASE BALANCE
 The normal blood pH is slightly alkaline (pH 7.35-7.45)
and the body through its metabolic processes produce
more acids than bases, hence, there must be a
mechanism that would maintain the slight alkalinity of
the blood.
 The kidney functions to eliminate the acids and
conserve the bases and this is accomplished by:
a. Reabsorption of filtered bicarbonates
b. Elimination of organic acids( i.e. lactic acid)
c. Secretion of ammonia by tubular cells
 ENDOCRINE FUNCTION

1. Secretion of Renal Erythropoietic Factor (REF)

 The juxta glomerular cells (JG) of the kidney
secretes REF which combines with a globulin coming
from the liver to produce erythropoietin, a hormone
essential for the maintenance of normal RBC
production.
HYPOXIA

Kidney liver
REF globulin
Erythropoietin erythropoieses
 ENDOCRINE FUNCTION
 2. Production of renin
 Aside from secreting REF, the juxta-glomerular cells also

secrete renin which is implicated in the regulation of osmotic

equilibrium and ionic balance in the plasma (Renin-
Angiotensisn-Aldosterone System)
3. Activation of Vitamin D
the formation of 1,25-dihydroxycholecalciferol occurs in the
kidneys.
inactive Vit.D3 kidneys active Vit.d#
THREE MAJOR GROUPS OF KIDNEY FUNCTION
TESTS

1. Tests measuring the Glomerular

Filtration Rate (GFR)
2. Tests measuring Renal Blood Flow
3. Tests measuring Tubular Function
 TESTS MEASURING THE
GLOMERULAR FILTRATION RATE
(GFR)
 Tests which measure the rate of glomerular filtration are generally called
CLEARANCES. These group of tests (renal clearance tests) are useful in
measuring the actual excretory capacity of the kidneys since they measure the
amount of substance excreted in the urine, as compared to the concentration of
the same substance in plasma.
 ESTIMATION OF GLOMERULAR FILTRATION:
 How much of plasma that courses through the kidneys is filtered per minute?
This could be calculated if we have substances having the following essential
characteristics.
a. Freely filtered or allowed to pass through the filtering apparatus
b. Neither secreted nor reabsorbed from the filtrate as it courses along nephric
tubules.
c. Neither synthesized nor altered by the kidneys (not metabolized)
d. Easily detected or analyzed chemically in both plasma and urine.
 GENERAL FORMULA FOR CLEARANCE
 C (mL/min) = U x volume (mL/min) x 1.73
P A
Where : C – clearance of the substance expressed in mL/min
U – concentration of substance in urine
P – concentration of substance in plasma
Volume (mL/min) – total volume of urine excreted in 24 hours converted to
mL/min.
1.73 - generally accepted body surface area of an individual in square
meters
A - body surface area of patient whose value is obtained from a
nomogram (height and weight are needed)
  Types of Clearance Tests
 Creatinine Clearance Test
 Inulin Clearance Test
 Urea Clearance Test
 INULIN CLEARANCE TEST
 - (gold standard)- inulin freely passes the glomeruli but is neither
secreted or reabsorbed by the nephric tubules. Since inulin is not present
in the plasma, it must be introduced in a suitable concentration in order
to allow its clearance by the kidneys to be measured. Thus, considered to
be the most accurate measure of GFR.
 This is done by giving a priming dose (25ml of 10% inulin solution) by

intravenous injection to produce a satisfactory plasma level and then

maintaining this level throughout the test period by a slow, continuous
intravenous infusion of a less concentrated solution (5oomL inulin
solution)
 In actual clinical setting, this is not commonly requested due to lack of
feasible procedure. It is therefore not regarded as a regular clearance
test.
 Normal value: 125ml/min(men due to larger renal mass), 110ml/min-

women
INULIN CLEARANCE TESTS
 consideredto be the most accurate
measure of GFR
 Normal value: 250 ml/min. - men

(due to larger renal mass)

110 mL/min - women
 Creatinine Clearance Test

 Creatinine is the end product of muscle metabolism derived

from creatine. It is freely filtered by the glomeruli but not
reabsorbed by the tubules. However, it is secreted by the
renal tubules resulting in an overesimation of the actual
GFR. Creatinine is the most commonly used endogenous
substance in the clinical asessment of GFR.
 Creatinine occuring through metabolic production is
eliminated from the plasma by glomerular filtration and
therefore a measurement of its rate of clearance affords a
measure of the process.
 Normal value: 107-139ml/min (men) and 87-107 ml/min
(women)

UREA CLEARANCE TEST
Urea is freely filtered by the glomeruli but is variably
reabsorbed in the tubules depending upon the transit
time (rate of urine flow along the courseof nephric
tubules) of the urea filtrate. The faster the rate of urine
flow, the less urea is reabsorbed; the slower the rate of
urine flow, the more urea is reabsorbed.
 A. if the rate of urine flow is 2 ml/min or more, less urea
is reabsorbed and the average normal clearance is
75ml/min (64-99ml/min) . This is known as the
Maximum Urea clearance.
 UREA CLEARANCE TEST
 Formula: Cm (ml/min) = U x Volume (ml/min) x 1.33 x 1.73
 P A
 1.33 - constant for maximum urea clearance
 B. if the rate of urine flow is less than 2 ml/min, more
urea is reabsorbed and the average normal clearance is
54 ml/min (41-68 ml/min). This is known as the Standard
Urea Clearance
 Formula: Cs (ml/min) = U x √ vol (ml/min) x 1.85 x 1.73
 P A

 1.85 - constant for standard urea clearance

 TESTS MEASURING RENAL BLOOD FLOW
 The Non-Protein Nitrogenous (NPN) substances are waste products of
metabolism , removed from the blood stream by the kidneys. The most
important of which, according to descending order of nitrogen contributions
are urea, amino acid, uric acid, creatinine, creatine and ammonia.
 The total NPN determination is not done frequently in clinical laboratories
because of the following:
1. Method of deproteinization of blood or serum may qualitatively or
quantitatively alter NPN compounds:
a. TCA filtrate retain all NPN compounds
b. Somogyi filtrates adsorb some NPN compounds
2. Not all NPN substances are specific for renal failures:
a. In cases of muscular diseases, creatinine and creatine are elevated.
b. In liver disease, ammonia and urea concentration may also be elevated.

NON-PROTEIN NITROGEN
6 major components
 Urea – major: 45-50% of total NPN
 End product of protein metabolism

 Amino acids – 20%

 Creatine
 Creatinine – waste product of muscle catabolism
 Ammonia – from deamination of amino acids; associated
with Reye’s syndrome
 Uric Acid – end product of purine/NA metabolism; for
the diagnosis of gout
TESTS FOR NPN
 Urea determination
 90% is excreted in the urine
 Major organic solid in the urine
 Creatinine determination
 Almost 100% excreted
 Ratio of urea to creatinine; 10:1
 More sensitive index for kidney function
 Uric Acid determination
 Cannot be used alone for kidney function because only
10% is excreted in the kidney
METHODS OF DETERMINATION OF NPN

A. DIGESTION PROCEDURE
 Kjeldahl digestion process
 Principle:
The nitrogen ion in the PFF of the specimen
in converted to ammonia using hot
concentrated sulfuric acid in the presence of a
catalyst (copper sulfate, selenium oxide or
mercuric- sulfate)
 N2 conc.H2 SO4 NH3
CuSO4
selenium oxide
mercuric sulfate
METHODS OF DETERMINATION OF NPN

B. MEASUREMENT OF AMMONIA
The amount of ammonia NH3 formed in the kjedahl digestion
process can be measured by either:
 1. Nesslerization
Principle:
The ammonia formed in reacted with the Nessler's
reagent (double iodide of potassium and mercury, K2Hg212)
In the presence of a colloidal stabilizer (gum ghatti) to form a
colloidal suspension of dimercuric ammonium iodide
(NH2Hg2l2), which is said to be yellow in color, if nitrogen is
present in low to moderate concentration, and orange brown,
if nitrogen is present in high concentration
NH3 + K2Hg2I2 gum ghatti NH2Hg2I2
(colloidal stabilizer)
METHODS OF DETERMINATION
B. MEASUREMENT OF AMMONIA
 2. BERTHELOT'S REACTION
 Principle:
 The ammonia formed is reacted with phenol and

alkaline hypochlorite using sodium nitroprusside as

catalyst to form indophenol blue. It is said to be more
specific than nesslerization.
 NH3 + phenol + alkaline hypochlorite
 sodium nitroprusside
 (catalysts)
 indophenol blue
 BLOOD UREA NITROGEN (BUN)

 Urea is the principal waste of protein catabolism. It is formed in the

liver in a process called urea cycle, thus in severe liver damage,
levels of urea decreases. It is also the first metabolite to increase in
kidney disease, hence, used as a screening test for such. Urea is
readily removed by dialysis.
 BUN determination:
 Principle: hydrolysis of urea by urease
 The urea in the sample is hydrolyzed by a specific enzyme,
urease, to form ammonia and ammonium carbonate. The
amount of ammonia formed can be measured by (1)
Nesslerization, (2) Berthelot reaction and (3) Acid
Titration.
 METHODS OF BUN
 1. Enzymatic and Nesslerization
 Urea in the PFF is made to react with urease glycerol
extract in the presence of buffer and heat-forming
ammoonium carbonate which is then reacted with
Nessler’s reagent to form a yellow dimercuric ammonium
iodide. The intensity of the color is directly proportional to
the amount of nitrogen in the sample.
i. Urease
ii. Modified kaar
iii. Gentzkov base
iv. Folin Svedberg
2. Enzymatic and Berthelot’s Reaction
urea is hydrolyzed to ammonium carbonate by urease and
then ammonia reacts with phenol and sodium hypochlorite
in an alkaline medium forming a blue indophenol.
v. Berthelot
 METHODS OF BUN
 3. Enzymatic and Acid Titration
Urea is hydrolyzed by urease forming ammonia which is then
titrated with a weak acid.
VI. Van Slyke Gullen
VII. Urograph
UROGRAPH METHOD- Principle: Paper Chromatography-
physical
Conway Microdiffusion- chemical
Strip used: Urostrat Chromatography paper strip
indicator system (BCG + Tartaric acid)
plastic barrier (stop the diffusion)

potassium carbonate
urease
When urea in serum comes in contact with
urease by diffusion, ammonium carbonate is formed w/c reacts with
potassium carbonate to release ammonia. The ammonia released
reacts with the dye and titrated by the acid to form a blue green
color. The amount of nitrogen present is determined by the height of
the color which measured after 30 minutes standing at room
temperature. It is measured by:
  A.) ruler- height in nm x 5 + 10 = BUN in mg% x 0.357 =
mmol/L
 B.) caliper - direct reading in mg% x 0.357 = mmol/L
 To convert BUN to urea , multiply BUN by 2.14
 IV. Direct Measurement of UREA
• urea is not hydrolyzed to ammonia but is measured
directly.
• VIII. DIACETYL MONOXIME METHOD (DAM)
 PRINCIPLE: Urea is made to react with diacetyl

monoxime to produce a yellow diazine derivative

(Fearon’s reaction) .Arsenic thiosemicarbazide is
added to enhance color formation and to exclude
protein interference.
 Disadvatages: lack of specificity, instability of
color produced, deviation from Beer’s Law and
toxicity & irritating odor of reagent.
METHODS OF DETERMINATION FOR BUN
 Use the factor 2.14 [BUN x 2.14 = Urea]
 Old Method
 Micro-Kjeldahl Nessler
 Most Popular Method
 DAM (diacetyl monoxime)
 Urea + DAM = orange-yellow compound
 Fearon Method, Rosenthal Method, Autoanalyzer method
 Urease Nessler Method
 Urea → CO2 + NH3
 NH3 + Nessler’s rgt → NH2HGI3
 Urease Berthelot Method
1.
C ONSIDERATIONS
The INbyBUN
determination is affected DETERMINATION
high protein diet, hydration and other
physiologic functions.
2. Whole blood should be deproteinized to eliminate interferences of
hemoglobin.
3. Ammonia containing anticoagulants are contraindicated in enzymatic
methods.
4. Sodium fluoride inhibits the action of urease.
5. Upon prolonged standing, ammonia concentration in the sample rises 2-
3 times the original value due to enzymatic deamination of labile amides
like glutamine.
NORMAL VALUE: 7-8 mg/dL (2.5 – 8.4 mmol/L)
CONVERSION FACTOR: 0.357
CLINICAL SIGNIFICANCE:
 BUN determination is almost always requested
with creatinine since they appear to aid in the
differential diagnosis of pre-renal, renal and post-
renal hyperuremia
 Pre-renal causes - conditions in which circulation through
the kidneys is less efficient than usual.
 Hemorrhage (blood loss)
 Cardiac decompression
 Increased protein catabolism
 Heatstroke (Dehydration)
 Burns (Fluid loss)
CLINICAL SIGNIFICANCE:

 Renal causes - characterized by the presence of lesions on the

parenchyma itself (tubular injury).
 Chronic nephritis
 Acute glomerulonephritis (AGN)
 Polycystic kidney
 Nephroschlerosis
 Tubular necrosis
 Post-renal causes - due to the obstruction in the-urinary-tract
due to:
 Stones
 Prostatic enlargement
 Tumors
CLINICAL SIGNIFICANCE:
 AZOTEMIA - ("azo”) - nitrogen containing - biochemical
abnormality that refers to an increase in BUN and
creatinine levels which is largely related to decrease
glomerular filtration.
 UREMIA - defined as the increased in urea and creatinine
(azotemia) with accompanying clinical signs and symptoms
of renal failure like:
 Metabolic acidosis due to failure of the kidneys to
eliminate acidic products of metabolism
 Hyperkalemia due to failure of potassium excretion
 Generalized edema due to water retention
 CREATININE
 Creatinine is the principal waste product of muscular metabolism derived
mainly from creatine (alpha-methyl guanidoacetic acid). It is synthesized
from three amino acids (methionine, arginine and lysine.) in the skeletal
muscles, it is phosphorylated to phosphoryl creatine(phosphocreatine)
catalyzed by the enzyme creatine phosphokinase (crteatine kinase).
Phosphoryl creatine provides the immediate source of ATP during muscle
contraction.
 Creatinine is cleared from the plasma by glomerular filtration and this is
excreted in the urine without being reabsorbed by the tubules. Its excretion
is relatively constant, hence, creatinine output is sometimes measured as a
check for the completeness of 24 hour urine sample collection. It increases in
the latter part of renal diseases thus offering a more reliable screening test
for renal pathology and as an index of over-all renal function. It is not
readily removed by dialysis and also not affected by protein diet except for
canned sardines. Not elevated unless 25-50% of the nephrons destroyed(
1kidney equivalent)
 CREATININE DETERMINATION
METHODS:
 1. ENZYMATIC METHODS
 CREATINASE METHOD
• PRINCIPLE: Available on Ektachem wherein creatinine
is hydrolyzed to N-methylhydantoin and ammonia by
creatinase. The ammonia is then made to react with
alpha-ketoglutarate and NADH in the presence of
glumate dehydrogenase forming glutamate and NAD. The
decrease in NADH is followed fluoremetrically.
CREATININE AMINOHYDROLASE METHOD
PRINCIPLE: Creatinine is hydrolyzed to creatine by
creatinine aminhydrolase, followed by a series of coupled-
enzyme reactions in w/c creatine reacts with creatinine
kinase, pyruvate kinase and lactate dehydrogenase,
culminating in the oxidation of NADH.
II. Direct Jaffe ‘s Reaction Method
Principle: ( Jaffe’ reaction) It is the formation of
red tautomer of creatinine picrate when
creatinine in serum is made to react with a
freshly prepared alkaline sodium picrate solution
(alkaline picrate- - Jaffe reagent)
Compositon of the Jaffe reagent
1 part of 10% NaOH
5 parts of saturated picric acid (2,4,6
trinitrophenol)
Alk. Picrate: picric acid dissolved in 10% NaOH
Should be freshly prepared to prevent its
conversion to picramic acid and methylguanidine
after 4 hrs (picramic acid)
 1. Folin Wu Method- a sensitive method but not
specific since it measures other
chromogenic substances like ascorbic acid, ketones
and barbiturates.
 2. Lloyd Method – a sensitive and specific method
since it measures “true” creatinine. It is the reference
method for creatinine. Its advantage is that
creatinine is adsorbed by the aluminum silicate or
oxalic acid (lloyds Reagent) which isolates creatinine
form other interfering substances. Elution techniques
are then utilized to separate the creatinine from the
adsorbent which is then made to react specifically
with the freshly prepared Jaffe reagent.
 Addition of Lloyd’s reagent (aluminum silicate) to
make Jaffe reaction specific to creatinine
 Creatinine + alk. Picrate →creatinine picrate (orange
red compound)
CREATININE
 NORMAL VALUE: 0.6 - 1.2 mg/dL (53-106 umol/L)
 CONVERSION FACTOR: 88.4
 CLINICAL SIGNIFICANCE:
 Aside from renal diseases, it is also elevated in myopathies
like:
a. Muscular dystrophies
b. Familial periodic paralysis
c. Myasthenia gravis
d. Dermatomyositis
 BLOOD URIC ACID (BUA)
 Uric acid is the major end product of purine
metabolism formed in the liver and intestinal mucosa
from xanthine by the action of xanthine oxidase. In
humans, uric acid is chiefly excreted in the urine. It is
filtered by the glomerulus, reabsorbed and secreted by
the tubules. Normally , 98% of the filtered UA is
reabsorbed. It is relatively insoluble, thus, when it
accumulates, it may be deposited in the joints (tophi) or
in the genitourinary tracts as uric acid stones.
 Cannot be used alone to evaluate kidney function; 10%
is excreted in the kidney
 BUA DETERMINATION
 I. Direct Redox Methods
 Principle: Uric acid is oxidized to allantoin and CO2 by phosphotungstic
acid reagent (protein precipitant and color rgt.) in alkaline solution. In
the process the phosphotungstic acid is reduced to tungsten blue.
 uric acid + phosphotungstic acid
 sodium cyanide or sodium carbonate
 tungsten blue + allantoin + CO2
 Methods based on their alkalinizing agent:
i. Sodium cyanide (NaCN)- Folin, Brown, Newton and Benedict
ii. Sodium carbonate (Na2CO3) – Henry, Caraway and Archibald
Lag phase- incubation period after the addition of an alkali (NaCN or
Na2CO3) to inactivate non-uric acid reactants primarily ascorbic acid and
polyphenols that would elevate uric acids levels.
 Caraway Method
UA + PTA → Tungsten Blue
* Uses NaCN as color stabilizer
 Modification of Caraway (Henry’s method)

UA + PTA → Tungsten Blue

* Uses Na2CO3 as color stabilizer
 Autoanalyzer Method

Uricase method
UA →absorbance reading @ 290-293 nm
UA + Uricase → allantoin
Allantoin →absorbance reading @ 290-293 nm
 BUA
 Considerations in BUA determination:
1. Uric acid is stable for several days at room temperature and
longer if refrigerated.
2. Addition of thymol increases its stability to bacterial
destruction.
3. Any of the common anticoagulants can be used except for
potassium oxalate which forms potassium phosphotungstate
promoting turbidity
4. Purine like foods like legumes, visceral organs and others may
affect uric acid assay.
 NORMAL VALUES: 4.0 - 8.5 mg/dL(0.25 - 0.50 mmol/L) –
men
2.7 - 7.3 mg/dl (0.16 - 0.43 mmol/L)
- women
 CONVERSION FACTOR: 0.059
CLINICAL SIGINIFCANCE:
 Gout - a defect in uric acid metabolism which causes an excess
of the acid and salts to accumulate in the bloodstream and
joints.
 Chronic alcoholism increases uric acid in the bloodstream
because alcohol inhibits its excretion.
 Leukemia and other malignant conditions due to increased
turnover of nucleoproteins.
 Uric acid levels are elevated in decreased renal functions
either due to over production of uric acid or decreased rate.of
excretion.
 Fatal poisoning with chloroform and methanol, excessive
exposure to X-rays and radioactive radiators, due to excessive
cell breakdown and nucleic acid catabolism
 Genetic disease such as: Lesch-Nyhan syndrome and Von
Glerk's disease
 AMMONIA
 ammonia determinations contribute very little or none at
all on renal impairment. It has its significance in
impending hepatic coma and terminal stages of hepatic
cirrhosis.
AMINO ACIDS
Quantitative amino acid determinations are important in
congenital renal disorder wherein aminoaciduria results.
CREATINE
Synthesized from amino acids arginine, methionine and
glycerine. It is increased in muscular dystrophies.
 TESTS MEASURING TUBULAR FUNCTIONS

 A. EXCRETORY TESTS:
 1. Para- Amino Hippurate Test (PAH) or
Diodrast Test
 2. Phenolsulfonphthalein (PSP) Dye

Excretion Test
B. CONCENTRATION TEST
1. Specific gravity

2. Osmolality

3. Fishberg Concentration Test

 Clinical
chemistry 1
Compilation of
Computations
SHIRLEY O.SOLITARIO,RMT
BASIC CHEMISTRY AND LAB
MATHEMATICS
 PERCENT SOLUTION
 A. weight/volume
 FORMULA:
% w/v= grams of solute/ mL of solution x 100
Where as:
grams of solute = % soln desired x total vol.desired
100
 PERCENT SOLUTION
 B. % volume per volume (v/v)
 FORMULA: % v/v = mL of solute x 100

 mL of solution
 Where as:

 mL of solute= % soln.desired x total vol. desired

 100
 PERCENT SOLUTION
C. % weight per weight (w/w)
Formula:
% w/w = grams of solute x 100
grams of solution
Where as:
Grams of solute= % soln.desired x grams of total soln.
100
Note: When preparing concentrated acid
solutions, always add acid to water.
 THREE BASIC TYPES OF SOLUTIONS
 Molar Solutions (MOLARITY)
 solution containing one gram molecular weight (one mole of
the solute in one liter solution) of the substance per liter of
the solution
 The number of moles expressed per 1 liter of solution.
 It is expressed as moles per liter (mol/L)
 FORMULA:
 mole = grams of solute
 molecular weight (MW)
MOLARITY(M)= grams of solute
MW x volume of solution (L)
 CONVERSION OF % W/V TO M

To convert % w/v to Molarity

(M)
M = % w/v x 10
GMW
 NORMALITY

 FORMULAS:
 Equivalent weight (EW)= molecular weight

valence
 Normality (N) = grams of solution

equivalent weight x volume (L)

CONVERSION OF % W/V TO NORMALITY
 N= % w/v x10
 EW
 RELATIONSHIP BETWEEN MOLARITY AND
NORMALITY
 NORMALITY = MOLARITY x VALENCE
 MOLARITY = NORMALITY / VALENCE

 NORMALITY IS ALWAYS EQUAL TO OR

GREATER THAN MOLARITY.
 MOLARITY IS ALWAYS EQUAL TO OR LESS
THAN NORMALITY
 MOLALITY
 Amount of solute per 1kg of solvent
 It is expressed as moles per kilogram (mol/kg) or
weight/weight.
 Molecular weight (MW) is obtained by adding the
atomic weight of the given compound.
 FORMULA:

 Molality (m)= grams of solute

 MW x kilogram of solvent
 MILLIEQUIVALENTS
 The most common way of expressing electrolytes
 A milliequivalent is the equivalent weight
expressed in milligrams.
 Typically, the laboratory is required to convert
milligrams per deciliter (mg/dL) to
milliequivalent per liter (mEq/L)
 FORMULA:

 mEq/L = mg/dL x 10 x valence

 molecular weight (MW)
 MILLIMOLES
 Millimoles is the molecular weight expressed in
milligrams. (mmol/L)
 Formula:
 mmol/L = mg/dL x 10
 MW
 Example Problem:
 1. Convert a 3mg/dL magnesium to mmol/L
 MW= 24.31
 mmol/L = 3 x10/ 24.31= 1.23 mmol/L
 2. Convert 8.2 mg/dL calcium to mmol/L.
 MW= 40
 mmol/L = 8.2 x 10 /40 = 2.05mmol/L
 DILUTION
 Amount of solute (A) = Volume A x conc,A
 Amount of solute (B) = Volume B x conc.B

 Hence:
 Volume A x conc.A = Volume B x conc.B
 N1 x V1 = N2 x V2
 Sample poblem: Prepare 1Liter of 0.1 N HCl(N2) from 12.1N
concentrated HCl (N1) calculate the amount (in mL) of
conc.HCl(V1) to dilute with distilled water (V2)
 N1 V1 = N2 V2
 12.1 x V1 = 0.1 x 1000
 x = 100/12.1 = 8.26 mL
 V1 (x) = 8.26 mL of concentrated HCl to be diluted to 1 liter
distilled water.
 RATIO
 VOLUME OF SOLUTE PER VOLUME OF
SOLVENT

 RATIO = volume of solute

 volume of solvent
QUALITY
CONTROL
FORMULA 2: CALCULATING A STANDARD
DEVIATION [S] FOR A SET OF QC VALUES
VARIANCE

LIPIDS
Low density lipoprotein
Determined using the Friedewald equation:
TC = HDL + LDL + VLDL
Hence:
LDL = TC – (HDL + TG/5) if TG <400
LDL = TC – (HDL + TG x 0.16) if TG >400
LDL = TC – (HDL + TG/2.175) if TG is in mmol/L
General formula:
LDL = TC- HDL- VLDL
 VLDL CHOLESTEROL

 Friedewald

VLDL= trigly = mg/dL

5

VLDL = trigly = mmol/L

2.175
 VLDL DETERMINATIONS

DE LONG
VLDL= trigly = mg/dL
6.5

VLDL = trigly = mmol/L

2.825
 DETERMINATION OF HDL
CHOLESTEROL
Precipitating reagents such
as divalent cations and
polyanions are used to
remove all lipoproteins
except HDL.
Enzymatic method for total
cholesterol is used to
quantitate HDL.
PROTEINS
DETERMINATION OF INDIVIDUAL OF
PROTEINS

1. Total Protein =Albumin + globulin

Globulin =TP – albumin
A/G ratio = albumin/globulin :1
A/G ratio = answer :1
inverted if globulin is greater than albumin
A/G ratio = 1: answer
 Albumin-Globulin Ratio
 1.5-3.5/1

 A/G RATIO: If albumin is greater than

globulin
 A/G ratio= Albumin/Globulin :1
If globulin is greater than albumin
A/G ratio = 1: globulin/albumin
 Inverted A/G ratio – seen in multiple
myeloma
DETERMINATION OF INDIVIDUAL
OPROTEINS

2. GLOBULIN
- total globulins are measured by subtracting albumin from the total protein
concentration:

TP – albumin = globulin
Normal values:
Conventional SI unit
Total protein 6.0-7.8 g/dL 6078 g/L
Albumin 3.2-4.5 g/dL 32-45 g/L
Globulin 2.3-3.5 g/dL 23-35 g/L

Conversion factor: 10
DETERMINATION OF INDIVIDUAL
OPROTEINS
3.FIBRINOGEN
- most abundant of coagulation factors responsible for the formation of fibrin
clot
- only protein found in plasma which is not found in serum
FORMULA:
TP (PLASMA) - TP (SERUM) = FIBRINOGEN
METHODS OF DETERMINATION:
Parfentjev method
Fibrinogen is precipitated with ammonium sulphate and sodium chloride
from a citrated plasma. A dilution of mixture is then read
spectrophotometrically at 510 nm.
Howe’s method
Fibrinogen is precipitated with calcium chloride and assayed using the
Kjeldahl method.
NORMAL VALUE: 200-400 mg/dL (2.0-4.0 g/L)
KFT
 GENERAL FORMULA FOR CLEARANCE

 C (mL/min) = U x volume (mL/min) x 1.73

P A
Where : C – clearance of the substance expressed in mL/min
U – concentration of substance in urine
P – concentration of substance in plasma
Volume (mL/min) – total volume of urine excreted in 24 hours converted to
mL/min.
1.73 - generally accepted body surface area of an individual in square
meters
A - body surface area of patient whose value is obtained from a
nomogram (height and weight are needed)
 UREA CLEARANCE TEST
 Formula: Cm (ml/min) = U x Volume (ml/min) x
1.33 x 1.73
P A
 1.33 - constant for maximum urea clearance

 B. if the rate of urine flow is less than 2 ml/min, more urea

is reabsorbed and the average normal clearance is 54
ml/min (41-68 ml/min). This is known as the Standard
Urea Clearance
 Formula: Cs (ml/min) = U x √ vol (ml/min) x 1.85 x
1.73
P
A
 1.85 - constant for standard urea clearance
METHODS OF DETERMINATION FOR BUN
 Usethe factor 2.14
 BUN x 2.14 = Urea